Peptides for diagnostic and therapeutic methods for celiac sprue

ABSTRACT

Detection of toxic gluten oligopeptides refractory to digestion and antibodies and T cells responsive thereto can be used to diagnose Celiac Sprue. Analogs of such oligopeptides are useful in the inhibition of immune responses.

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes disease insensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

Celiac Sprue is generally considered to be an autoimmune disease and theantibodies found in the serum of the patients support a theory of animmunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active CS, and the presence of such antibodies, particularly of theIgA class, has been used in diagnosis of the disease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villous atrophy of the small intestine.

At the present time there is no good therapy for the disease, except tocompletely avoid all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. An important cause of death islymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for examples in commercial soups, sauces, icecreams, hot dogs, etc., that patients need detailed lists of foodstuffsto avoid and expert advice from a dietitian familiar with celiacdisease. Ingesting even small amounts of gluten may prevent remission orinduce relapse. Supplementary vitamins, minerals, and hematinics mayalso be required, depending on deficiency. A few patients respond poorlyor not at all to gluten withdrawal, either because the diagnosis isincorrect or because the disease is refractory. In the latter case, oralcorticosteroids (e.g., prednisone 10 to 20 mg bid) may induce response.

Current diagnostic methods for Celiac Sprue are expensive and not veryaccurate. These methods include ELISA-based methods in which eitheranti-gliadin or anti-tTG antibodies in the patient's serum are detectedand in which T cell proliferation upon stimulation with gliadin isobserved. Often, however, these methods are not sensitive enough todetect the diagnostic antibodies in the blood or, as is the case for Tcell proliferation assays, are deemed to be too expensive for routineuse. Typically, even if an individual tests positive in the diagnostictest, the individual must be re-challenged with gliadin (typically aftermaintaining a gluten-free diet for an extended period of time) andexamined by endoscopy, an invasive and often painful procedure.

PCT publication No. WO 01/25793, published 12 Apr. 2001, describespeptides derived from epitope mapping of alpha-gliadin and methods fordiagnosing Celiac Sprue using such peptides. Those methods, however, donot appear to be significantly more sensitive than methods currentlyemployed and so do not overcome the limitations of diagnostic methodscurrently in use.

PCT publication No. WO 02/083722 describes HLA-DQ restricted T cellsreceptors capable of recognizing prolamine-derived peptides involved infood-related immune enteropathy.

There remains a need for better diagnostic methods for Celiac Sprue,methods that are more sensitive than current methods, that do notrequire confirmation by endoscopy, and that do not require that anindividual be challenged with a gluten-containing diet for accuracy. Thepresent invention addresses this need.

SUMMARY OF THE INVENTION

Methods are provided for diagnosing Celiac Sprue, and/or dermatitisherpetiformis, by detecting multivalent toxic gluten oligopeptides in apatient; antibodies that bind to the toxic gluten oligopeptides; or Tcell proliferation elicited by such oligopeptides in a patient. Novelpeptides are provided, which interact strongly with gluten reactive Tcells and/or HLA molecules. Certain peptides, particularly modifiedpeptides, are shown to bind strongly to the HLA molecule, withoutactivating T cells, thereby blocking reactivity. Such peptides find usein diagnostic and therapeutic methods.

In one aspect, the present invention provides methods for treatingCeliac Sprue and/or dermatitis herpetiformis and the symptoms thereof byadministration of an HLA-binding peptide inhibitor to the patient. Inone embodiment, the HLA-binding peptide inhibitor employed in the methodis an analog of an immunogenic gluten peptide, where an immunogenicgluten peptide is altered by the replacement of one or more amino acids,where the replacement may be another naturally occurring amino acid,non-naturally occurring amino acids, modified amino acids, amino acidmimetics, and the like. Analogs of immunogenic gluten peptides that (i)retain the ability to bind tightly to HLA molecules; (ii) retain theproteolytic stability of these peptides; but (iii) are unable toactivate disease-specific or other T cells, are useful agents to treatCeliac Sprue.

In another aspect, the present invention provides novel HLA-bindingpeptide inhibitors and methods for treating Celiac Sprue and/ordermatitis herpetiformis by administering those compounds.

In another aspect, the invention provides pharmaceutical formulationscomprising an HLA-binding peptide inhibitor and a pharmaceuticallyacceptable carrier. In one embodiment, such formulations comprise anenteric coating that allows delivery of the active agent to theintestine, and the agents are stabilized to resist digestion oracid-catalyzed modification in acidic stomach conditions. In anotherembodiment, the formulation also comprises one or more glutenases, asdescribed in U.S. Provisional Application 60/392,782 filed Jun. 28,2002; and U.S. Provisional Application 60/428,033, filed Nov. 20, 2002,both of which are incorporated herein by reference. The invention alsoprovides methods for the administration of enteric formulations of oneor more HLA-binding peptide inhibitors to treat Celiac Sprue.

These and other aspects and embodiments of the invention and methods formaking and using the invention are described in more detail in thedescription of the drawings and the invention, the examples, the claims,and the drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Brush border membrane catalyzed digestion of theimmunodominant gliadin peptide. FIG. 1A: LC-MS traces of (SEQ ID NO:1)QLQPFPQPQLPY after digestion with 27 ng/μl rat brush border membrane(BBM) protein for the indicated time. Reaction products were separatedby reversed phase HPLC and detected by mass spectroscopy (ion countsm/z=300-2000 g/mol). The indicated peptide fragments were confirmed bycharacteristic tandem MS fragmentation patterns. The SEQ ID NO:2pyroQLQPFPQPQLPY peak corresponds to an N-terminally pyroglutaminatedspecies, which is generated during HPLC purification of the syntheticstarting material. FIG. 1B Abundance of individual digestion products asa function of time. The peptide fragments in FIG. 1A were quantified byintegrating the corresponding MS peak area (m/z=300-2000 g/mol). Theresulting MS intensities are plotted as a function of digestion time(with BBM only, colored bars). The digestion experiment was repeated inthe presence of exogenous DPP IV from Aspergillus fumigatus (ChemiconInternational, CA, 0.28 μU DPP IV/ng BBM protein) and analyzed as above(open bars). The relative abundance of different intermediates could beestimated from the UV₂₈₀ traces and control experiments using authenticstandards. The inserted scheme shows an interpretative diagram of thedigestion pathways of (SEQ ID NO:1) QLQPFPQPQLPY and its intermediates,the BBM peptidases involved in each step, and the amino acid residuesthat are released. The color code for labeling the peptides is similarto that used in A. The preferred breakdown pathway is indicated in bold.APN=aminopeptidase N, CPP=carboxypeptidase P, DPP IV=dipeptidyldipeptidase IV.

FIG. 2A-2B. C-terminal digestion of the immunodominant gliadin peptideby brush border membrane. FIG. 2A: (SEQ ID NO:3) PQPQLPYPQPQLPY wasdigested by 27 ng/μl brush border membrane (BBM) protein preparationsfor the indicated time and analyzed as in FIG. 1A. The identity of thestarting material and the product (SEQ ID NO:4) PQPQLPYPQPQLP wascorroborated by MSMS fragmentation. The intrinsic mass intensities ofthe two peptides were identical, and the UV₂₈₀ extinction coefficient of(SEQ ID NO:4) PQPQLPYPQPQLP was half of the starting material inaccordance with the loss of one tyrosine. All other intermediates werebelow ≦1%. The scheme below shows the proposed BBM digestion pathway of(SEQ ID NO:3) PQPQLPYPQPQLPY with no observed N-terminal processing(crossed arrow) and the removal of the C-terminal tyrosine bycarboxypeptidase P (CPP) in bold. Further C-terminal processing bydipeptidyl carboxypeptidase (DCP) was too slow to permit analysis of thesubsequent digestion steps (dotted arrows). FIG. 2B: Influence ofdipeptidyl carboxypeptidase on C-terminal digestion. (SEQ ID NO:3)PQPQLPYPQPQLPY in phosphate buffered saline:Tris buffered saline=9:1 wasdigested by BBM alone or with addition of exogenous rabbit lung DCP(Cortex Biochemicals, CA) or captopril. After overnight incubation, thefraction of accumulated SEQ ID NO:4) PQPQLPYPQPQLP (compared to initialamounts of (SEQ ID NO:3) PQPQLPYPQPQLPY at t=0 min) was analyzed as inFIG. 2A, but with an acetonitrile gradient of 20-65% in 6-35 minutes.

FIG. 3. Dose dependent acceleration of brush border mediated digestionby exogenous endoproteases. As seen from FIG. 2A-2B, the peptide (SEQ IDNO:4) PQPQLPYPQPQLP is stable toward further digestion. This peptide wasdigested with 27 ng/μl brush border membranes, either alone, withincreasing amounts of exogenous prolyl endopeptidase (PEP, specificactivity 28 μU/pg) from Flavobacterium meningosepticum (US Biological,MA), or with additional elastase (E-1250, Sigma, Mo.), bromelain(B-5144, Sigma, Mo.) or papain (P-5306, Sigma, Mo.). After one hour, thefraction of remaining (SEQ ID NO:4) PQPQLPYPQPQLP (compared to theinitial amount at t=0 min) was analyzed and quantified as in FIG. 1.

FIG. 4. Products of gastric and pancreatic protease mediated digestionof α2-gliadin under physiological conditions. Analysis was performed byLC-MS. The longest peptides are highlighted by arrows and also in thesequence of α2-gliadin (inset).

FIG. 5. In vivo brush border membrane digestion of peptides. LC-UV₂₁₅traces of 25 μM of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFbefore perfusion and after perfusion (residence time=20 min). LC-UV₂₁₅traces of 50 μM of (SEQ ID NO:1) QLQPFPQPQLPY before perfusion and afterperfusion (residence time=20 min).

FIG. 6. Alignment of representative gluten and non-gluten peptideshomologous to (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

FIG. 7. Breakdown and detoxification of 33-mer gliadin peptide with PEP.In vitro incubation of PEP (540 mU/ml) with the 33-mer gliadin peptide(100 μM) for the indicated time. In vivo digestion of the 33-mer gliadinpeptide (25 μM) with PEP (25 mU/ml) and the rat's intestine (residencetime=20 min).

FIG. 8. Equilibrium occupancy of individual peptides shown in FIG. 10 inthe DQ2 binding pocket, as measured by peptide exchange assays.Measurements were made at (A) pH 5.5 and (B) pH 7.3. 4.7 μM DQ2 wasmixed with 0.18 μM fluorescein-conjugated peptide at 37° C. for 45 h,and the abundance of DQ2 bound peptide was calculated as a percentage oftotal peptide.

FIG. 9A-9B. Stimulation of T cell proliferation by three peptides, the33-mer 1 (Δ), peptide 3 ( ), and the 20-mer 5 (⋄).Paraformaldehyde-fixed DQ2 cells were used as antigen presenting cells.(A) Proliferation of a polyclonal T cell line that recognizes all aepitopes. (B) Proliferation of a clonal T cell line that recognizes theαII epitope (peptide 3).

FIG. 10. Structures of candidate DQ2 blocking agents 19-22.

FIG. 11A-11C. Kinetic analysis of exchange of compounds 17-21 in the DQ2binding pocket. (A) Exchange of compounds 17 (Δ) and 19 ( ) onto DQ2 atpH 5.5 (filled circles) and pH 7.3 (open circles). (B) Exchange ofcompounds 18 (Δ) and 20 ( ) onto DQ2 at pH 5.5 (filled circles) and pH7.3 (open circles). (C) Comparative kinetics of DQ2 binding of compounds5 ( ), 19 (Δ), 20 (∇), and 21 (⋄) at pH 5.5 and 37° C.

FIG. 12. Kinetic analysis of exchange of compound 22 (filled) and 33-mer1 (filled Δ) at pH 5.5. Data at pH 7.3 was similar; at this pH peptide 1reaches a maximum occupancy of 28% (45 h), whereas peptide 22 reaches amaximum occupancy of 40% (20 h).

FIG. 13A-13B. Comparison of T cell proliferation in the presence ofmodified peptides 19, 20, 21, and 22. All peptides were tested at aconcentration of 3 μM, except compound 22 was tested at 5 μM. DQ2 APCswere γ-irradiated before incubation with peptide.

FIG. 14. T cell response to various concentrations of antigen peptide 5co-incubated without any blocker peptide or in the presence of compound21 or compound 22 (5 μM each) as reversible inhibitors of DQ2 antigenpresentation. DQ2 APCs were fixed prior to incubation with peptide.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are diagnosed by detectingantibodies that bind to digestion refractory gluten oligopeptides and/orT-cell proliferation produced by such oligopeptides in Celiac Sprueindividuals. Gluten oligopeptides are highly resistant to cleavage bygastric and pancreatic peptidases such as pepsin, trypsin, chymotrypsin,and the like.-By providing for detection of such gluten oligopeptides;of antibodies specifically reactive thereto; and/or of T-cellproliferation produced by such oligopeptides in individuals, improvedmethods of diagnosing Celiac Sprue and/or dermatitis herpetiformis areprovided.

Celiac Sprue and/or dermatitis herpetiformis may also be treated byinterfering with HLA binding of immunogenic gluten peptides. Therapeuticbenefit can also be enhanced in some individuals by increasing thedigestion of gluten oligopeptides, whether by pretreatment of foodstuffsto be ingested or by administration of an enzyme capable of digestingthe gluten oligopeptides, together with administration of an HLA-bindingpeptide inhibitor. Gluten oligopeptides are highly resistant to cleavageby gastric and pancreatic peptidases such as pepsin, trypsin,chymotrypsin, and the like, and their prolonged presence in thedigestive tract can induce an autoimmune response. The antigenicity ofgluten oligopeptides and the ill effects caused by an immune responsethereto can be decreased by administration of an HLA-binding peptideinhibitor. Such inhibitors are analogs of immunogenic gluten peptidesand (i) retain the ability to bind tightly to HLA molecules; (ii) retainthe proteolytic stability of these peptides; but (iii) are unable toactivate disease-specific or other T cells.

In some embodiments and for some individuals, the methods of theinvention remove the requirement that abstention from ingestion ofglutens be maintained to keep the disease in remission. The compositionsof the invention include formulations of tTGase inhibitors that comprisean enteric coating that allows delivery of the agents to the intestinein an active form; the agents are stabilized to resist digestion oralternative chemical transformations in acidic stomach conditions. Inanother embodiment, food is pretreated or combined with glutenase, or aglutenase is co-administered (whether in time or in a formulation of theinvention) with an HLA-binding peptide inhibitor of the invention.

The subject methods are useful for both prophylactic and therapeuticpurposes. Thus, as used herein, the term “treating” is used to refer toboth prevention of disease, and treatment of a pre-existing condition.The treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient, is a particularly important benefit provided bythe present invention. Such treatment is desirably performed prior toloss of function in the affected tissues; consequently, the prophylactictherapeutic benefits provided by the invention are also important.Evidence of therapeutic effect may be any diminution in the severity ofdisease, particularly diminution of the severity of such symptoms asfatigue, chronic diarrhea, malabsorption of nutrients, weight loss,abdominal distension, and anemia. Other disease indicia include thepresence of antibodies specific for glutens, antibodies specific fortissue transglutaminase, the presence of pro-inflammatory T cells andcytokines, and degradation of the villus structure of the smallintestine. Application of the methods and compositions of the inventioncan result in the improvement of any and all of these disease indicia ofCeliac Sprue.

Patients that can benefit from the present invention include both adultsand children. Children in particular benefit from prophylactictreatment, as prevention of early exposure to toxic gluten peptides canprevent development of the disease into its more severe forms. Childrensuitable for prophylaxis in accordance with the methods of the inventioncan be identified by genetic testing for predisposition, e.g. by HLAtyping; by family history, and by other methods known in the art. As isknown in the art for other medications, and in accordance with theteachings herein, dosages of the HLA-binding peptide inhibitors of theinvention can be adjusted for pediatric use.

Because most proteases and peptidases are unable to hydrolyze the amidebonds of proline residues, the abundance of proline residues in gliadinsand related proteins from wheat, rye and barley can constitute a majordigestive obstacle for the enzymes involved. This leads to an increasedconcentration of relatively stable gluten derived oligopeptides in thegut. These stable gluten derived oligopeptides, called “immunogenicoligopeptides” herein, bind to MHC molecules, including HLA HLA-DQ2 orDQ8 molecules, to stimulate an immune response that results in theautoimmune disease aspects of Celiac Sprue. In some cases the enzymetissue transglutaminase selectively deamidates certain glutamineresidues in these peptides, thereby enhancing their potency for the DQ2ligand binding pocket.

Peptides of particular interest for these purposes are analogs of SEQ IDNO:12 LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. Such analogs may comprise oneor more of the modifications set forth herein. Analogs may be truncatedof 5, 10, 12, 13 or more amino acids, which truncation may be from theamino or the carboxy terminus. Analogs may be deamidated at one, two,three or more glutamine residues, by substitution with glutamic acid atthese positions, where deamidation of residues 10, 17 and 24 are ofparticular interest. Analogs may be substituted at one, two three ormore leucine residues for lysine, where substitution of positions 11 and18 are of particular interest; and/or modification of the substitutedlysine residues with a sterically hindered conjugate to the ε-aminegroup.

Deamidated, and in some instances truncated, analogs of SEQ ID NO:12include:

ID Number SEQ ID NO:12 1 LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF SEQ ID NO:172     PFPQPELPY SEQ ID NO:18 3       PQPELPYPQ SEQ ID NO:19 4LQLQPFPQPELPYPQ SEQ ID NO:20 5 LQLQPFPQPELPYPQPELPY SEQ ID NO:21 6      PQPELPYPQPELPY SEQ ID NO:22 7       PQPELPYPQPELPYPQPELPY SEQ IDNO:23 8     PFPQPELPYPQPELPYPQPELPYPQPQP SEQ ID NO:24 9  LQPFPQPELPYPQPELPYPQPELPYPQPQP SEQ ID NO:25 10 QLQPFPQPELPYPQPELPYPQPELPYPQPQP SEQ ID NO:26 11LQLQPFPQPELPYPQPQLPYPQPQLPYPQPQPF SEQ ID NO:27 12LQLQPFPQPQLPYPQPELPYPQPQLPYPQPQPF SEQ ID NO:28 13LQLQPFPQPQLPYPQPQLPYPQPELPYPQPQPF SEQ ID NO:29 14LQLQPFPQPELPYPQPELPYPQPQLPYPQPQPF SEQ ID NO:30 15LQLQPFPQPELPYPQPQLPYPQPELPYPQPQPF SEQ ID NO:31 16LQLQPFPQPQLPYPQPELPYPQPELPYPQPQPF

It can be seen from these sequences that analogs comprising variouscombinations of deamidated residues can be produced (SEQ ID NO:17-23),and will bind to the HLA molecule DQ2. Additional modifications includedeletion of terminal residues, for example SEQ ID NO:24-SEQ ID NO:30.The antigenic response of the 33mer is principally centered around the(SEQ ID NO:31) (SEQ ID NO: 18) PQPELPYPQ epitope (residues 7-15), butthat the N-terminal sequence (SEQ ID NO:32) LQLQPF as well as aC-terminally located secondary Glu (e.g. E17 or E24) residue furtherenhance DQ2 affinity of the 33mer.

In one embodiment of the invention, a peptide of at least about 14 aminoacids, at least 18 amino acids, at least 20 amino acids, at least 22amino acids, and not more than about 33 amino acids, not more than about28, not more than about 26, or not more than about 24 amino acids isprovided, wherein said peptide comprises the epitope sequence (SEQ IDNO: 18) PQPELPYPQ. The peptide may optionally comprise as the aminoterminal sequence, (SEQ ID NO:32), LQLQPF. For example, such a peptidemay comprise the sequence of SEQ ID NO:27, and may further comprise aC-terminally located secondary glu residue. In some embodiments, thecarboxy terminal sequence is SEQ ID NO:44 PELPY, or SEQ ID NO:45 PEKPY,e.g. (SEQ ID NO:34), LQLQPFPQPELPYPQPEKPY

In another embodiment, the peptide of interest described above comprisesthe epitope sequence (SEQ ID NO:43) PQPEKPYPQ, wherein the leucine hasbeen replaced with a lysine residue. Such peptides may optionallycomprise as the amino terminal sequence, SEQ ID NO:32, LQLQPF, forexample SEQ ID NO:33 LQLQPFPQPEKPYPQPELPY. Such peptide may optionallycomprise a C-terminally located secondary glu residue. In someembodiments, the carboxy terminal sequence is SEQ ID NO:44 PELPY, or SEQID NO:45 PEKPY, for example SEQ ID NO:35 LQLQPFPQPEKPYPQPEKPY.

The ε-amine of the lysine is generally reactive to electrophiles, andcan readily by modified. The lysine residues in the peptides describedabove may be conjugated to a group that provides for steric hindrance ofinteractions between the peptide and a cognate receptor. Such conjugatesmay include, without limitation, succinic acid; glutaric acid;γ-aminobutyric acid; benzyloxycarbonyl group; t-Butoxycarbonyl group;9-fluorenylmethoxycarbonyl group; phthalimides; polyethylene glycol;secondary and tertiary amines. Peptides thus modified have been found tobind very well to HLA antigens, but do not activate T cells thatproliferate in response to SEQ ID NO:12. Such peptides therefore finduse as inhibitors of immune responses involved in Celiac Sprue and/ordermatitis herpetiformis.

Peptides as described above, including, without limitation, thosecomprising or consisting of SEQ ID NO:43, SEQ ID NO:33, SEQ ID NO:35,may additionally be substituted with cysteine. Sites for cysteinesubstitution include the sites of lysine substitution (residue 11 and 18with respect to SEQ ID NOL35). Such analogs are transformed into DQ2blockers through intramolecular cyclization via disulfide bondformation. Cyclic DQ2 binding molecules may further be modified byaltering the bridge lengths, e.g. replacing cysteine with analogues suchas homocysteine. More stable analogues are prepared by replacingdisulfide bonds with other flexible cyclization tethers. Two strategiesof interest are replacement of disulfide bridges with thioether linkages[Robey, F. A. (2000) Selective and Facile Cyclization ofNchloroacetylated Peptides from the C4 Domain of HIV Gp120 in LiCl/DMFSolvent Systems. J. Peptide Res. 56, 115-120; Oligino, L.; Lung, F.-D.T.; Sastry, L.; Bigelow, J.; Cao, T.; Curran, M.; Burke, T. R., Jr.;Wang, S.; Krag, D.; Roller, P. P.; King, C. R. (1997) NonphosphorylatedPeptide Ligands for the Grb2 Src Homology 2 Domain. J. Biol. Chem. 272,29046-29052] and olefin metathesis [Blackwell, H. E.; Grubbs, R. H.(1998) Highly Efficient Synthesis of Covalently Cross-linked PeptideHelices by Ring-Closing Metathesis. Angew. Chem. Int. Ed. 37, 3281-3284;Schafmeister C. E.; Po, J.; Verdine, G. L. (2000) An All-HydrocarbonCross-Linking System for Enhancing the Helicity and Metabolic Stabilityof Peptides. J. Am. Chem. Soc. 122, 5891-5892]. Alternatively, a widerange of bis-alkylating agents such as dibromoketones are used, sincethe resulting macrocyclic products, if active, will contain anorthogonal ketone functional group for further modification

In another embodiment, an immunogenic gluten oligopeptide analog is ananalog of a peptide that comprises at least about 8 residues, and maycomprise at least about 10 residues; at least about 11 residues, atleast about 12 residues, at least about 13 residues, at least about 14residues, or more, where the term “residue” refers to naturallyoccurring amino acids, non-naturally occurring amino acids, and aminoacid mimetics or derivatives; and where the gluten peptide is altered bythe replacement of one or more amino acids. The replacement may beanother naturally occurring amino acid, non-naturally occurring aminoacids, modified amino acids, amino acid mimetics, and the like; and mayfurther be derivitized to further reduce the affinity of these ligandsfor disease-specific T cell receptors. The sequence of immunogenicgluten oligopeptides can be determined by one of skill in the art.Immunogenic gliadin oligopeptides are peptides derived during normalhuman digestion of gliadins and related storage proteins as describedabove, from dietary cereals, e.g. wheat, rye, barley, and the like. Sucholigopeptides act as antigens for T cells in Celiac Sprue. For bindingto Class II MHC proteins, immunogenic peptides are usually from about 8to 20 amino acids in length, more usually from about 10 to 18 aminoacids. Such peptides may include PXP motifs, such as the motif (SEQ IDNO: 8) PQPQLP. Determination of whether an oligopeptide is immunogenicfor a particular patient is readily determined by standard T cellactivation and other assays known to those of skill in the art.

Among gluten proteins with potential harmful effect to Celiac Spruepatients are included the storage proteins of wheat, species of whichinclude Triticum aestivum; Triticum aethiopicum; Triticum baeoticum;Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticumtimopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii;Triticum zhukovskyi; etc. A review of the genes encoding wheat storageproteins may be found in Colot (1990) Genet Eng (N Y) 12:225-41. Gliadinis the alcohol-soluble protein fraction of wheat gluten. Gliadins aretypically rich in glutamine and proline, particularly in the N-terminalpart. For example, the first 100 amino acids of α- and γ-gliadinscontain ˜35% and ˜20% of glutamine and proline residues, respectively.Many wheat gliadins have been characterized, and as there are manystrains of wheat and other cereals, it is anticipated that many moresequences will be identified using routine methods of molecular biology.Examples of gliadin sequences include but are not limited to wheat alphagliadin sequences, for example as provided in Genbank, accession numbersAJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606;AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306;U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin isset forth in Genbank accession number AF280605.

Among the immunogenic gluten oligopeptides that may be modified togenerate an HLA-binding peptide inhibitor are included the peptidesequence (SEQ ID NO:44) QLQPFPQPELPYP; the sequence (SEQ ID NO:36)PQPELPY; the sequence (SEQ ID NO:46) PFPQPELPYP, (SEQ ID NO:47)PQPELPYPQPQLP, (SEQ ID NO:48) PQQSFPEQQPP, (SEQ ID NO:49)VQGQGIIQPEQPAQ, (SEQ ID NO:50) FPEQPQQPYPQQP, (SEQ ID NO:51)FPQQPEQPYPQQP, (SEQ ID NO:52) FSQPEQEFPQPQ and longer peptidescontaining such sequences or multiple copies of such sequences.Gliadins, secalins and hordeins contain several (SEQ ID NO:53) PQPQLPYsequences or sequences similar thereto rich in Pro-Gln residues that arehigh-affinity substrates for tTGase. The tTGase catalyzed deamidation ofsuch sequences increases their affinity for HLA-DQ2, the class II MHCallele present in >90% Celiac Sprue patients. Presentation of thesedeamidated sequences by DQ2 positive antigen presenting cellseffectively stimulates proliferation of gliadin-specific T cells fromintestinal biopsies of most Celiac Sprue patients, providing evidencefor the proposed mechanism of disease progression in Celiac Sprue.

Analog oligopeptides of the invention comprise at least one differencein amino acid sequence from a native gluten peptide, by the replacementof an amino acid with a different amino acid; a non-naturally occurringamino acid, a peptidomimetics, substituted amino acid, and the like. AnL-amino acid from the native peptide may be altered to any other one ofthe 20 L-amino acids commonly found in proteins, any one of thecorresponding D-amino acids, rare amino acids, such as 4-hydroxyproline,and hydroxylysine, or a non-protein amino acid, such as β-alanine,ornithine and homoserine. Also included with the scope of the presentinvention are amino acids that have been altered by chemical means suchas methylation (e.g., α-methylvaline), deamidation, amidation of theC-terminal amino acid by an alkylamine such as ethylamine, ethanolamine,and ethylene diamine, and acylation or methylation of an amino acid sidechain function (e.g., acylation of the epsilon amino group of lysine),deimination of arginine to citrulline, isoaspartylation, orphosphorylation on serine, threonine, tyrosine or histidine residues.Importantly, each of these altered amino acids provide a functionalhandle, e.g. amine, alcohol, aryl halide, and the like, which can beregioselectively derivatized to further reduce the affinity of theseligands for disease-specific T cell receptors. Peptide analogs may befurther derivatized with substitutions, including, without limitation,ethers, amines, esters, amides, carbonates, carbamates, carbazates,ureas and C—C coupled derivatives. Other examples include oxidation ofalcohols to ketones, followed by further modifications of the resultingcarbonyl group, e.g. via preparation of oximes) or the carbon atomadjacent to the ketone. Such derivatives are encompassed by the term“analog”.

The proteolytic stability of gluten oligopeptides can be attributed, atleast in part, to the presence of PXP motifs, which are resistant toenzymatic degradation. Preferred analogs of immunogenic glutenoligopeptides will comprise one or more proline residues, and maycomprise one or more PXP motifs.

One inhibitor of interest is an oligopeptide or peptidomimetic thatcomprises the sequence PXPQPELPY, where X is Gly, Ala, Tyr, Trp, Arg,Lys, p-iodo-Phe, 3-iodo-Tyr, p-amino-Phe, 3-amino-Tyr, hydroxylysine,ornithine, Asp, Glu, or any residue that is substantially bulkier orhydrophilic than Phe. Examples of suitable modifications include ethers,amines, esters, amides, carbonates, carbamates, carbazates, ureas andC—C coupled derivatives. Other examples include oxidation of alcohols toketones, followed by further modifications of the resulting carbonylgroup (e.g. via preparation of oximes) or the carbon atom adjacent tothe ketone. The peptide may comprise modifications that increase bindingpotency to an MHC molecule, by varying residues that facilitate peptidedocking into the binding cleft. Examples of such residues include Gln-4,Glu-6, Leu-7, and Tyr-9 (numbering based on the epitope (SEQ ID NO: 17)PFPQPELPY). Each of these residues interacts closely with severalresidues in the DQ2 binding pocket. By using structure-based moleculardesign methods, these interactions can be optimized.

Another inhibitor of interest is a oligopeptide or peptidomimetic thatcomprises the sequence PFPQX₁ELX₂Y, where X₁ and X₂ are independentlyselected from 4-hydroxy-Pro (either isomer at C-4), 4-amino-Pro (eitherisomer atC-4), or 3-hydroxy-Pro (either isomer atC-3), and proline, withthe proviso that at least one of X₁ and X₂ is a residue other thanproline.

As described above, the sequence of gluten peptides may be altered invarious ways known in the art to generate targeted changes in sequence.The sequence changes may be substitutions, insertions or deletions. Suchalterations may be used to alter properties of the protein, by affectingthe stability, specificity, etc. Techniques for in vitro mutagenesis ofcloned genes are known. Examples of protocols for scanning mutations maybe found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene37:111-23 (1985); Colicelli et al., Mol Gen Genet. 199:537-9 (1985); andPrentki et al., Gene 29:303-13 (1984). Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene126:35-41 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jonesand Winistorfer, Biotechniques 12:528-30 (1992); Barton et al., NucleicAcids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70(1989); and Zhu Anal Biochem 177:120-4 (1989).

The peptides may be joined to a wide variety of other oligopeptides orproteins for a variety of purposes. By providing for expression of thesubject peptides, various post-expression modifications may be achieved.For example, by employing the appropriate coding sequences, one mayprovide farnesylation or prenylation. The peptides may be PEGylated,where the polyethyleneoxy group provides for enhanced lifetime in theblood stream. The peptides may also be combined with other proteins,such as the Fc of an IgG isotype, which may be complement binding, witha toxin, such as ricin, abrin, diphtheria toxin, or the like, or withspecific binding agents that allow targeting to specific moieties on atarget cell.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, acylation,carboxylation, etc. Also embraced are sequences that have phosphorylatedamino acid residues, e.g. phosphotyrosine, phosphoserine, orphosphothreonine.

Also included in the subject invention are peptides that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradationor to optimize solubility properties or to render them more suitable asa therapeutic agent. Analogs of such polypeptides include thosecontaining residues other than naturally occurring L-amino acids, e.g.D-amino acids or non-naturally occurring synthetic amino acids. D-aminoacids may be substituted for some or all of the amino acid residues.

Peptides and peptide analogs may be synthesized by standard chemistrytechniques, including synthesis by automated procedure. In general,peptide analogs are prepared by solid-phase peptide synthesismethodology which involves coupling each protected amino acid residue toa resin support, preferably a 4-methylbenzhydrylamine resin, byactivation with dicyclohexylcarbodiimide to yield a peptide with aC-terminal amide. Alternatively, a chloromethyl resin (Merrifield resin)may be used to yield a peptide with a free carboxylic acid at theC-terminus. After the last residue has been attached, the protectedpeptide-resin is treated with hydrogen fluoride to cleave the peptidefrom the resin, as well as deprotect the side chain functional groups.Crude product can be further purified by gel filtration, HPLC, partitionchromatography, or ion-exchange chromatography.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

In one embodiment of the invention, the peptide consists essentially ofa polypeptide sequence as set forth in any one of the SEQ ID NOsprovided herein. By “consisting essentially of” in the context of apolypeptide described herein, it is meant that the polypeptide iscomposed of the gluten sequence, which sequence may be flanked by one ormore amino acid or other residues that do not materially affect thebasic characteristic(s) of the polypeptide.

Interactions with Immune System Receptors

Preferably, antigenic oligopeptides of interest for use in the methodsof the invention are as described above, and comprise at least oneepitope. As used herein, the term “epitope” refers to the portion of anantigen bound by an antibody or T cell receptor, which portion issufficient for high affinity binding. In polypeptide antigens, generallya linear epitope for recognition will be at least about 7 amino acids inlength, and may be 8 amino acids, 9 amino acids, 10 amino acids, ormore.

Antibodies may recognize linear determinants or conformationaldeterminants formed by non-contiguous residues on an antigen, and anepitope can therefore require a larger fragment of the antigen to bepresent for binding, e.g. a protein domain, or substantially all of aprotein sequence. The binding site of antibodies typically utilizesmultiple non-covalent interactions to achieve high affinity binding.While a few contact residues of the antigen may be brought into closeproximity to the binding pocket, other parts of the antigen molecule canalso be required for maintaining a conformation that permits binding. Inorder to consider an antibody interaction to be “specific”, the affinitywill be at least about 10⁻⁷ M, usually about 10⁻⁸ M to 10⁻⁹ M, and maybe up to 10⁻¹¹ M or higher for the epitope of interest. It will beunderstood by those of skill in the art that the term “specificity”refers to such a high affinity binding, and is not intended to mean thatthe antibody cannot bind to other molecules as well. One may findcross-reactivity with different epitopes, due, e.g. to a relatedness ofantigen sequence or structure, or to the structure of the antibodybinding pocket itself.

The T cell receptor recognizes a more complex structure than antibodies,and requires both a major histocompatibility antigen binding pocket andan antigenic peptide to be present. The binding affinity of T cellreceptors is lower than that of antibodies, and will usually be at leastabout 10⁻⁴ M, more usually at least about 10⁻⁵ M.

Affinity and stability are different measures of binding interaction.The definition of affinity is a thermodynamic expression of the strengthof interaction between a single antigen binding site and a singleantigenic determinant (and thus of the stereochemical compatibilitybetween them). Affinity does not change with valency, because it is themeasure of interaction between a single binding site and a singleantigenic determinant. In contrast to affinity, avidity (which relatesto the t_(1/2) of an interaction) is defined as the strength of binding,usually of a small molecule with multiple binding sites by a largermolecule, and in particular, the binding of a complex antigen by anantibody. Therefore, it is avidity that takes into account the effect ofmultiple interactions, and it is the change in avidity that may providesthe hyperantigenicity observed with the oligopeptide of SEQ ID NO:12.

Certain of the gluten oligopeptides analogs described herein are usefulin stimulating T cells from Celiac Sprue patients for diagnosticpurposes, while others are shown to inhibit T cell stimulation. Suchpeptides are provided by the present invention in isolated and highlypurified forms. Further, the gluten oligopeptides analogs describedherein are useful in diagnostic assays for detecting antibodies againstsuch oligopeptides or for producing antibodies that bind specifically tosuch oligopeptides for their detection.

Diagnostic Methods

The present invention provides a variety of methods for diagnosingCeliac Sprue. In one embodiment, the diagnosis involves detecting thepresence of a gluten oligopeptides digestion product, e.g. SEQ ID NO:12;deamidated counterparts there; a tTGase-linked counterpart thereof;etc., in a tissue, bodily fluid, or stool of an individual. Thedetecting step can be accomplished by use of a reagent, e.g. anantibody, that recognizes the indicated antigen, or by a cell thatproliferates in the presence of the indicated antigen and suitableantigen presenting cells, wherein said antigen presenting cells arecompatible with the MHC type of the proliferating cell, e.g. allogeneiccells, autologous cells, etc.

In another embodiment, the diagnosis involves detecting the presence ofan antibody, one or more T cells reactive with the 33-mer or adeamidated counterpart thereof, or a tTGase-linked counterpart thereofin a tissue, bodily fluid, or stool of an individual. In one embodiment,an antibody is detected by, for example, an agglutination assay using anantigen provided by the present invention. In another embodiment, a Tcell is detected by its proliferation in response to exposure to amultivalent gluten oligopeptide provided by the present invention andpresented by autologous or suitable allogeneic antigen presenting cells.

In one aspect, the methods and reagents of the present invention arecapable of detecting the toxic oligopeptides of gluten proteins ofwheat, barley, oats and rye remaining after digestion or partialdigestion of the same by a Celiac Sprue individual. Gluten is theprotein fraction in cereal dough, which can be subdivided into gluteninsand prolamines, which can be further subclassified as gliadins,secalins, hordeins, avenins from wheat, rye, barley and oat,respectively. For further discussion of gluten proteins, see the reviewby Wieser (1996) Acta Paediatr Suppl. 412:3-9; herein incorporated byreference. Among gluten proteins of interest are included the storageproteins of wheat, species of which include Triticum aestivum; Triticumaethiopicum; Triticum baeoticum; Triticum militinae; Triticummonococcum; Triticum sinskajae; Triticum timopheevii; Triticum turgidum;Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; and the like.A review of the genes encoding wheat storage proteins may be found inColot (1990) Genet Eng (N Y) 12:225-41.

Of particular interest is gliadin, which is the alcohol-soluble proteinfraction of wheat gluten. Gliadins are typically rich in glutamine andproline, particularly in the N-terminal part. For example, the first 100amino acids of α- and γ-gliadins contain ˜35% and ˜20% of glutamine andproline residues, respectively. Many wheat gliadins have beencharacterized, and as there are many strains of wheat and other cereals,it is anticipated that many more sequences will be obtained usingroutine methods of molecular biology. Examples of sequenced gliadinsinclude wheat alpha gliadin sequences, for example as provided inGenbank, accession numbers AJ133612; AJ133611; AJ133610; AJ133609;AJ133608; AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602;D84341.1; U51307; U51306; U51304; U51303; U50984; and U08287. A sequenceof wheat omega gliadin is set forth in Genbank accession numberAF280605.

For the purposes of the present invention, toxic gliadin oligopeptidesare peptides derived during normal digestion of gliadins and relatedstorage proteins as described above, from dietary cereals, e.g. wheat,rye, barley, and the like, by a Celiac Sprue individual. Sucholigopeptides are believed to act as antigens for T cells in CeliacSprue individuals. For binding to Class II MHC proteins, immunogenicpeptides are usually from about 6 to 20 amino acids in length, moreusually from about 10 to 18 amino acids, and as demonstrated herein, aparticularly stimulatory toxic gliadin oligopeptide is the multivalent33-mer described above. Such peptides include PXP motifs, for examplethe motif PQPQLP (SEQ ID NO:8). Determination of whether an oligopeptideis immunogenic for a particular patient is readily determined bystandard T cell activation assays known to those of skill in the art.Illustrative toxic gliadin oligopeptides of the invention are describedin Examples 1 and 2 below. The 33-mer gliadin oligopeptide of Example 2and its deamidated counterpart formed by tTGase are preferred toxicgliadin oligopeptides of the invention.

Samples may be obtained from patient tissue, which may be a mucosaltissue, including but not limited to oral, nasal, lung, and intestinalmucosal tissue, a bodily fluid, e.g. blood, sputum, urine, phlegm,lymph, and tears. One advantage of the present invention is that theantigens provided are such potent antigens, both for antibody-bindingand T-cell stimulation, that the diagnostic methods of the invention canbe employed with samples (tissue, bodily fluid, or stool) in which aCeliac Sprue diagnostic antibody, peptide, or T cell is present in verylow abundance. This allows the methods of the invention to be practicedin ways that are much less invasive, much less expensive, and much lessharmful to the Celiac Sprue individual.

Patients may be monitored for the presence of reactive T cells, usingone or more multivalent oligopeptides as described above. The presenceof such reactive T cells indicates the presence of an on-going immuneresponse. The antigen used in the assays is a gluten oligopeptide analogas described above; including, without limitation, SEQ ID NO:12;deamidated counterparts; tTGase fusions thereof; and derivatives.Cocktails comprising multiple oligopeptides; panels of peptides; etc.may be also used. Overlapping peptides may be generated, where eachpeptide is frameshifted from 1 to 5 amino acids, thereby generating aset of epitopes.

The diagnosis may determine the level of reactivity, e.g. based on thenumber of reactive T cells found in a sample, as compared to a negativecontrol from a naive host, or standardized to a data curve obtained fromone or more positive controls. In addition to detecting the qualitativeand quantitative presence of antigen reactive T cells, the T cells maybe typed as to the expression of cytokines known to increase or suppressinflammatory responses. While not necessary for diagnostic purposes, itmay also be desirable to type the epitopic specificity of the reactive Tcells, particularly for use in therapeutic administration of peptides.

T cells may be isolated from patient peripheral blood, lymph nodes,including peyer's patches and other gut-related lymph nodes, or fromtissue samples as described above. Reactivity assays may be performed onprimary T cells, or the cells may be fused to generate hybridomas. Suchreactive T cells may also be used for further analysis of diseaseprogression, by monitoring their in situ location, T cell receptorutilization, MHC cross-reactivity, etc. Assays for monitoring T cellresponsiveness are known in the art, and include proliferation assaysand cytokine release assays. Also of interest is an ELISA spot assay.

Proliferation assays measure the level of T cell proliferation inresponse to a specific antigen, and are widely used in the art. In onesuch assay, recipient lymph node, blood or spleen cells are obtained atone or more time points after transplantation. A suspension of fromabout 10⁴ to 10⁷ cells, usually from about 10⁵ to 10⁶ cells is preparedand washed, then cultured in the presence of a control antigen, and testantigens, as described above. The cells are usually cultured for severaldays. Antigen-induced proliferation is assessed by the monitoring thesynthesis of DNA by the cultures, e.g. incorporation of ³H-thymidineduring the last 18H of culture.

T cell cytotoxic assays measure the numbers of cytotoxic T cells havingspecificity for the test antigen. Lymphocytes are obtained at differenttime points after transplantation. Alloreactive cytotoxic T cells aretested for their ability to kill target cells bearing recipient MHCclass 1 molecules associated with peptides derived from a test antigen.In an exemplary assay, target cells presenting peptides from the testantigen, or a control antigen, are labeled with Na⁵¹CrO₄. The targetcells are then added to a suspension of candidate reactive lymphocytes.The cytotoxicity is measured by quantitating the release of Na⁵¹CrO₄from lysed cells. Controls for spontaneous and total release aretypically included in the assay. Percent specific ⁵¹Cr release may becalculated as follows: 100×(release by CTL−spontaneous release)/(totalrelease−spontaneous release).

Enzyme linked immunosorbent assay (ELISA) and ELISA spot assays are usedto determine the cytokine profile of reactive T cells, and may be usedto monitor for the expression of such cytokines as IL-2, IL-4, IL-5,γIFN, etc. The capture antibodies may be any antibody specific for acytokine of interest, where supernatants from the T cell proliferationassays, as described above, are conveniently used as a source ofantigen. After blocking and washing, labeled detector antibodies areadded, and the concentrations of protein present determined as afunction of the label that is bound.

In one embodiment of the invention, the presence of reactive T cells isdetermined by injecting a dose of the 33-mer peptide, or a derivative orfragment thereof as described above, subcutaneously or sub-mucosallyinto a patient, for example into the oral mucosa (see Lahteenoja et al.(2000) Am. J. Gastroenterology 95:2880, herein incorporated byreference). A control comprising medium alone, or an unrelated proteinis usually injected nearby at the same time. The site of injection isexamined after a period of time, by biopsy or for the presence of awheal.

A wheal at the site of injection is compared to that at the site of thecontrol injection, usually by measuring the size of the wheal. The skintest readings may be assessed by a variety of objective grading systems.A positive result for the presence of an immune response will show anincreased diameter at the site of polypeptide injection as compared tothe control.

Where a biopsy is performed, the specimen is examined for the presenceof increased numbers of immunologically active cells, e.g. T cells, Bcells, mast cells, and the like. For example, methods of histochemistryand/or immunohistochemistry may be used, as is known in the art. Thedensities of cells, including antigen specific T cells, mast cells, Bcells, etc. may be examined. It has been reported that increased numbersof intraepithelial CD8⁺ T cells may correlate with gliadin reactivity.

An alternative method relies on the detection of circulating antibodiesin a patient. Methods of detecting specific antibodies are well-known inthe art. Antibodies specific for multivalent gluten oligopeptides asdescribed above may be used in screening immunoassays. A sample is takenfrom the patient. Samples, as used herein, include biological fluidssuch as blood, tears, saliva, lymph, dialysis fluid and the like; organor tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. Blood samples and derivatives thereof are ofparticular interest.

Measuring the concentration of specific antibodies in a sample orfraction thereof may be accomplished by a variety of specific assays. Ingeneral, the assay will measure the reactivity between a patient sample,usually blood derived, generally in the form of plasma or serum. Thepatient sample may be used directly, or diluted as appropriate, usuallyabout 1:10 and usually not more than about 1:10,000. Immunoassays may beperformed in any physiological buffer, e.g. PBS, normal saline, HBSS,dPBS, etc.

In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first attaching the peptide to aninsoluble surface or support. The peptide may be bound to the surface byany convenient means, depending upon the nature of the surface, eitherdirectly or through specific antibodies. The particular manner ofbinding is not crucial so long as it is compatible with the reagents andoverall methods of the invention. They may be bound to the platescovalently or non-covalently, preferably non-covalently.

The insoluble supports may be any composition to which peptides can bebound, which is readily separated from soluble material, and which isotherwise compatible with the overall method of measuring antibodies.The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which thereceptor is bound include beads, e.g. magnetic beads, membranes andmicrotiter plates. These are typically made of glass, plastic (e.g.polystyrene), polysaccharides, nylon or nitrocellulose. Microtiterplates are especially convenient because a large number of assays can becarried out simultaneously, using small amounts of reagents and samples.

Before adding patient samples or fractions thereof, the non-specificbinding sites on the insoluble support i.e. those not occupied byantigen, are generally blocked. Preferred blocking agents includenon-interfering proteins such as bovine serum albumin, casein, gelatin,and the like. Alternatively, several detergents at non-interferingconcentrations, such as Tween, NP40, TX100, and the like may be used.

Samples, fractions or aliquots thereof are then added to separatelyassayable supports (for example, separate wells of a microtiter plate)containing support-bound antigenic peptide. Preferably, a series ofstandards, containing known concentrations of antibodies is assayed inparallel with the samples or aliquots thereof to serve as controls.

Generally from about 0.001 to 1 ml of sample, diluted or otherwise, issufficient, usually about 0.01 ml sufficing. Preferably, each sample andstandard will be added to multiple wells so that mean values can beobtained for each. The incubation time should be sufficient forantibodies molecules to bind the insoluble antigenic peptide. Generally,from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.

After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample.

After washing, a solution containing a second receptor specific for thepatient antibodies is applied. The receptor may be any compound thatbinds patient antibodies with sufficient specificity such that it can bedistinguished from other components present. In a preferred embodiment,second receptors are antibodies specific for patient antibodies, eithermonoclonal or polyclonal sera, e.g. mouse anti-human antibodies, mouseanti-dog antibodies, rabbit anti-cat antibodies, etc. Such second stageantibodies may be labeled to facilitate direct, or indirectquantification of binding. Examples of labels which permit directmeasurement of second receptor binding include radiolabels, such as ³Hor ¹²⁵I, fluorescers, dyes, beads, chemilumninescers, colloidalparticles, and the like. Examples of labels that permit indirectmeasurement of binding include enzymes where the substrate may providefor a colored or fluorescent product. In a preferred embodiment, thesecond receptors are antibodies labeled with a covalently bound enzymecapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. Alternatively, the second stage may beunlabeled, and a labeled third stage is used. Examples of secondreceptor/second receptor-specific molecule pairs includeantibody/anti-antibody and avidin (or streptavidin)/biotin. Since theresultant signal is thus amplified, this technique may be advantageouswhere only a small amount of antibodies is present.

After the second stage has bound, the insoluble support is generallyagain washed free of non-specifically bound molecules, and the signalproduced by the bound conjugate is detected by conventional means. Wherean enzyme conjugate is used, an appropriate enzyme substrate is providedso a detectable product is formed. More specifically, where a peroxidaseis the selected enzyme conjugate, a preferred substrate combination isH₂O₂ and is O-phenylenediamine, which yields a colored product underappropriate reaction conditions. Appropriate substrates for other enzymeconjugates such as those disclosed above are known to those skilled inthe art. Suitable reaction conditions as well as means for detecting thevarious useful conjugates or their products are also known to thoseskilled in the art. For the product of the substrate O-phenylenediaminefor example, light absorbance at 490-495 nm is conveniently measuredwith a spectrophotometer.

Generally the amount of bound antibodies detected will be compared tocontrol samples from normal patients. The presence of increased levelsof the antigen specific antibodies is indicative of disease, usually atleast about a 5 fold, 10 fold, or 100 fold increase will be taken as apositive reaction.

In some cases, a competitive assay will be used. In addition to thepatient sample, a competitor to the antibodies is added to the reactionmix. The competitor and the antibodies compete for binding to theantigenic peptide. Usually, the competitor molecule will be labeled anddetected as previously described, where the amount of competitor bindingwill be proportional to the amount of antibodies present. Theconcentration of competitor molecule will be from about 10 times themaximum anticipated antibodies concentration to about equalconcentration in order to make the most sensitive and linear range ofdetection.

An alternative protocol is to provide anti-patient antibodies bound tothe insoluble surface. After adding the sample and washing awaynon-specifically bound proteins, one or a combination of the testantigens are added, where the antigens are labeled, so as not tointerfere with binding to the antibodies. Conveniently, fused proteinsmay be employed, where the peptide sequence is fused to an enzymesequence, e.g. β-galactosidase.

It is particularly convenient in a clinical setting to perform theimmunoassay in a self-contained apparatus. A number of such methods areknown in the art. The apparatus will generally employ a continuousflow-path of a suitable filter or membrane, having at least threeregions, a fluid transport region, a sample region, and a measuringregion. The sample region is prevented from fluid transfer contact withthe other portions of the flow path prior to receiving the sample. Afterthe sample region receives the sample, it is brought into fluid transferrelationship with the other regions, and the fluid transfer regioncontacted with fluid to permit a reagent solution to pass through thesample region and into the measuring region. The measuring region mayhave bound to it the antigenic peptide, with a conjugate of an enzymewith an antibodies specific antibody employed as a reagent, generallyadded to the sample before application. Alternatively, the antigenicpeptide may be conjugated to an enzyme, with antibodies specificantibody bound to the measurement region.

Thus, in one aspect, the present invention provides a method fordiagnosing Celiac Sprue in an individual who has not consumed gluten foran extended period of time, such time including but not limited to oneday, one week, one month, and one year prior to the performance of thediagnostic method. The advantage conferred by this aspect of theinvention is that current diagnosis of a Celiac Sprue individualtypically involves a preliminary diagnosis, after which the individualis placed on a gluten-free diet. If the individual's symptoms abateafter initiation of the gluten-free diet, then the individual ischallenged with gluten, and another diagnostic test, such as anendoscopy or T cell proliferation assay, is performed to confirm thepreliminary diagnosis. This re-challenge with gluten causes extremediscomfort to the Celiac Sprue individual. One important benefitprovided by certain embodiments of the invention is that such are-challenge need not be performed to diagnose Celiac Sprue, becauseeven very low levels of 33-mer specific antibodies and T cell responderscan be identified using the methods of the invention.

In another aspect, the present invention provides a method fordiagnosing Celiac Sprue by detecting the presence of a 33-mer specificantibody or a T cell responder in a bodily tissue or fluid other thanintestinal mucosa. In this aspect of the invention, the diagnosticmethods are performed without recourse to endoscopy or intestinalbiopsy, thus avoiding the discomfort, pain, and expense attendant onsuch procedures in common use today.

The subject methods are useful not only for diagnosing Celiac Sprueindividuals but also for determining the efficacy of prophylactic ortherapeutic methods for Celiac Sprue as well as the efficacy of foodpreparation or treatment methods aimed at removing glutens or similarsubstances from food sources. Thus, a Celiac Sprue individualefficaciously treated with a prophylactic or therapeutic drug or othertherapy for Celiac Sprue tests more like a non-Celiac Sprue individualwith the methods of the invention. Likewise, the antibodies or T cellresponders, e.g. T cell lines, of the invention that detect the toxicgluten oligopeptides of the invention are useful in detecting gluten andgluten-like substances in food and so can be used to determine whether afood treated to remove such substances has been efficaciously treated.

As used herein, the term “treating” is used to refer to both preventionof disease, and treatment of pre-existing conditions. The treatment ofongoing disease, to stabilize or improve the clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to loss of function in the affected tissues. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly measuring the severity of such symptoms as fatigue, chronicdiarrhea, malabsorption of nutrients, weight loss, abdominal distension,and anemia. Other disease indicia include the presence of antibodiesspecific for the 33-mer of the invention or its deamidated counterparts,glutens, antibodies specific for tissue transglutaminase or tTGaselinked to the 33-mer of the invention or its deamidated counterparts,the presence of pro-inflammatory T cells and cytokines, histologicalexamination of the villus structure of the small intestine, and thelike. Patients may be adult or child, where children in particularbenefit from prophylactic treatment, as prevention of early exposure totoxic gluten peptides may prevent initial development of the disease.Children suitable for prophylaxis can be identified by genetic testingfor predisposition, e.g. by HLA typing; by family history, and,preferably, by the diagnostic methods of the present invention.

The various methods and reagents of the invention can be prepared andmodified as described below. Although specific methods and reagents areexemplified in the discussion below, it is understood that any of anumber of alternative methods, including those described above areequally applicable and suitable for use in practicing the invention. Itwill also be understood that an evaluation of the methods of theinvention may be carried out using procedures standard in the art,including the diagnostic and assessment methods described above.

The practice of the present invention may employ conventional techniquesof molecular biology (including recombinant techniques), microbiology,cell biology, biochemistry and immunology, which are within the scope ofthose of skill in the art. Such techniques are explained fully in theliterature, such as, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J.Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987);“Methods in Enzymology” (Academic Press, Inc.); “Handbook ofExperimental Immunology” (D. M. Weir & C. C. Blackwell, eds.); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction” (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991).

As noted above, the subject methods are useful to monitor the progressand efficacy of therapies to treat individuals suffering from CeliacSprue and/or dermatitis herpetiformis. Such therapies can involveadministration of an effective dose of glutenase and/or tTGaseinhibitor, through a pharmaceutical formulation, incorporating glutenaseinto food products, administering live organisms that express glutenase,and the like. As these therapies may not have been approved by the FDAor an equivalent other regulatory agency, the methods of the inventionhave application in clinical trials conducted to evaluate the safety andefficacy of such therapies. Diagnosis of suitable patients may utilize avariety of criteria known to those of skill in the art in addition tothose methods described herein. A quantitative increase in antibodiesspecific for gliadin, and/or tissue transglutaminase is indicative ofthe disease. Family histories and the presence of the HLA allelesHLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] areindicative of a susceptibility to the disease.

In addition to employing the diagnostic methods of the invention, thetherapeutic effect may be measured in terms of clinical outcome, or mayrely on immunological or biochemical tests. Suppression of thedeleterious T-cell activity can be measured by enumeration of reactiveTh1 cells, by quantitating the release of cytokines at the sites oflesions, or using other assays for the presence of autoimmune T cellsknown in the art. Alternatively, one may look for a reduction insymptoms of a disease.

Pharmaceutical Compositions

The HLA-binding peptide inhibitors are incorporated into a variety offormulations for therapeutic administration. In one aspect, the agentsare formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration can be achieved in various ways,usually by oral administration. The HLA-binding peptide inhibitors maybe systemic after administration or may be localized by virtue of theformulation, or by the use of an implant that acts to retain the activedose at the site of implantation.

In pharmaceutical dosage forms, the HLA-binding peptide inhibitors maybe administered in the form of their pharmaceutically acceptable salts,or they may also be used alone or in appropriate association, as well asin combination with other pharmaceutically active compounds. The agentsmay be combined, as previously described, to provide a cocktail ofactivities. The following methods and excipients are merely exemplaryand are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments, and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, and cellulose acetate phthalate.

Other enteric formulation comprise engineered polymer microspheres madeof biologically erodable polymers, which display strong adhesiveinteractions with gastrointestinal mucus and cellular linings, cantraverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71(3):307-18.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of glutenase calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular complex employedand the effect to be achieved, and the pharmacodynamics associated witheach complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Methods of Treatment

The subject methods are used to treat individuals suffering from CeliacSprue and/or dermatitis herpetiformis, by administering an effectivedose through a pharmaceutical formulation. Diagnosis of suitablepatients may utilize a variety of criteria known to those of skill inthe art. A quantitative increase in antibodies specific for gliadin,and/or tissue transglutaminase is indicative of the disease. Familyhistories and the presence of the HLA alleles HLA-DQ2 [DQ(a1*05, b1*02)]and/or DQ8 [DQ(a1*03, b1*0302)] are indicative of a susceptibility tothe disease. Specific peptide analogs may be administeredtherapeutically to decrease inflammation, and/or to induceantigen-specific tolerance to treat autoimmunity. Methods for thedelivery of peptides that are altered from a native peptide are known inthe art. Alteration of native peptides with selective changes of crucialresidues can induce unresponsiveness or change the responsiveness ofantigen-specific autoreactive T cells.

The therapeutic effect may be measured in terms of clinical outcome, ormay rely on immunological or biochemical tests. Suppression of thedeleterious T-cell activity can be measured by enumeration of reactiveTh1 cells, by quantitating the release of cytokines at the sites oflesions, or using other assays for the presence of autoimmune T cellsknown in the art. Alternatively, one may look for a reduction insymptoms of a disease.

Various methods for administration may be employed. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.Such treatment could either be before meals or on a once-a-day basis oron a once-a-week basis, depending on the half-life of the inhibitor. Atypical dose is at least about 1 μg, usually at least about 10 μg, moreusually at least about 0.1 mg, and not more than about 10 mg, usuallynot more than about 1 mg. Enteric coating of these peptides may alsoenhance their lifetimes in the gut, thereby permitting delivery to theproximal and distal small intestinal tissue. Treatment of otherautoimmune disorders such as Type I diabetes with such ligands mayinvolve oral, intravenous or intramuscular administration. The initialdose may be larger, followed by smaller maintenance doses. The dose maybe administered as infrequently as weekly or biweekly, or more oftenfractionated into smaller doses and administered daily, with meals,semi-weekly, etc. to maintain an effective dosage level.

The HLA-binding peptide inhibitors of the invention may be administeredin the treatment of Type 1 diabetes (IDDM). IDDM and celiac disease areboth immunologic disorders where specific HLA alleles are associatedwith disease risk. Transglutaminase autoantibodies can be found in somepatients with IDDM. The prevalence of transglutaminase autoantibodies ishigher in diabetic patients with HLA DQ2 or DQ8.

Human type I or insulin-dependent diabetes mellitus (IDDM) ischaracterized by autoimmune destruction of the β cells in the pancreaticislets of Langerhans. The depletion of β cells results in an inabilityto regulate levels of glucose in the blood. Overt diabetes occurs whenthe level of glucose in the blood rises above a specific level, usuallyabout 250 mg/dl. In humans a long presymptomatic period precedes theonset of diabetes. During this period there is a gradual loss ofpancreatic beta cell function. IDDM is currently treated by monitoringblood glucose levels to guide injection, or pump-based delivery, ofrecombinant insulin. Diet and exercise regimens contribute to achievingadequate blood glucose control. The inhibitors of the invention may beadministered alone, or in combination with other therapies. The route ofadministration may be oral, as described for treatment of Celiac Sprue,or may be injected, e.g. i.v., i.m., etc. Administration may beperformed during the pre-symptomatic phase, or in overt diabetes.

Related applications include U.S. Provisional application 60/357,238filed Feb. 14, 2002; to U.S. Provisional Application 60/380,761 filedMay 14, 2002; to U.S. Provisional Application 60/392,782 filed Jun. 28,2002; and U.S. Provisional application No. 60/422,933, filed Oct. 31,2002, each of which are herein specifically incorporated by reference.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Immunodominant Peptides of Gliadin are Protease Resistant

Recent studies have identified a small number of immunodominant peptidesfrom gliadin, which account for most of the stimulatory activity ofdietary gluten on intestinal and peripheral T lymphocytes found inCeliac patients. The proteolytic kinetics of these immunodominantpeptides were analyzed at the small intestinal surface. Using brushborder membrane vesicles from adult rat intestines, it was shown thatthese proline-glutamine-rich peptides are exceptionally resistant toenzymatic processing, and that dipeptidyl peptidase IV and dipeptidylcarboxypeptidase are the rate-limiting enzymes in their digestion. Theseresults support the conclusions drawn from the tests described inExample 2 that incomplete digestion of gliadin, which results in theformation of the 33-mer oligopeptide and its deamidated counterpartformed by tTGase action, contributes to the disease symptoms of CeliacSprue and can be employed in improved diagnostic methods for CeliacSprue.

To dissect this complex process, liquid chromatography coupled massspectroscopy analysis (LC-MS-MS) was utilized to investigate thepathways and associated kinetics of hydrolysis of immunodominant gliadinpeptides treated with rat BBM preparations. Because the rodent is anexcellent small animal model for human intestinal structure andfunction, rat BBM was chosen as a suitable model system for thesestudies.

BBM fractions were prepared from rat small intestinal mucosa asdescribed by Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35. Usingstandard assays, the specific activities of the known BB peptidases weredetermined to be 127 μU/μg for Aminopeptidase N (APN, EC 3.4.11.2), 60μU/μg for dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), and 41 μU/μgfor dipeptidyl carboxypeptidase (DCP, EC 3.4.15.1). No prolineaminopeptidase (EC 3.4.11.5) or prolyl endopeptidase activity (PEP, EC3.4.21.26) activity was detectable (<5 μU/μg). Alkaline phosphatase andsucrase were used as control BBM enzymes with activities of 66 μU/μg and350 μU/μg, respectively.

BBM fractions were partially purified from the small intestinal mucosaof adult female rats maintained on an ad libitum diet of wheat-basedstandard rodent chow. Total protein content was determined by a modifiedmethod of Lowry with BSA as a standard. Alkaline phosphatase activitywas determined with nitrophenyl phosphate. Sucrase activity was measuredusing a coupled glucose assay. DPP IV, proline aminopeptidase and APNwere assayed continuously at 30° C. in 0.1M Tris-HCl, pH 8.0, containing1 mM of the p-nitroanilides (ε=8,800 M⁻¹ cm⁻¹) Gly-Pro-pNA, Pro-pNA orLeu-pNA, the latter in additional 1% DMSO to improve solubility. DCPactivity was measured in a 100 μl reaction as the release of hippuricacid from Hip-His-Leu. PEP activity was determined continuously with 0.4mM Z-Gly-Pro-pNA in PBS:H₂O:dioxane (8:1.2:0.8) at 30° C. One unit isdefined as the consumption of 1 μmol substrate per minute.

DPP IV and DCP are both up-regulated by a high proline content in thediet. However, APN activity using standard substrates was found to behigher than DPP IV even when fed extreme proline rich diets. Also,although a higher DCP vs. CPP activity has been observed with the modelpeptide Z-GPLAP at saturating concentrations, a difference in Km valuescould easily account the reversed ratio measured in this study. 100 μMwas chosen as the initial peptide concentration, since non-saturatingkinetics (k_(cat)/K_(m)) were considered to be physiologically morerelevant than the maximal rates of hydrolysis (k_(cat)).

Proteolysis with the BBM preparation was investigated using the peptide(SEQ ID NO:1) (SEQ ID NO:1) QLQPFPQPQLPY, a product of chymotrypticdigestion of α-9 gliadin (Arentz-Hansen et al. (2000) J. Exp. Med. 191,603-12). This peptide has been shown to stimulate proliferation of Tcells isolated from most Celiac Sprue patients, and hence is consideredto possess an immunodominant epitope. It was subjected to BBM digestion,followed by LC-MS-MS analysis. A standard 50 μl digestion mixturecontained 100 μM of synthetic peptide, 10 μM tryptophan andCbz-tryptophan as internal standards, and resuspended BBM preparationswith a final protein content of 27 ng/μl and exogenous proteins, asindicated, in phosphate buffered saline. After incubation at 37° C. forthe indicated time, the enzymes were inactivated by heating to 95° C.for 3 minutes. The reaction mixtures were analyzed by LC-MS(SpectraSystem, ThermoFinnigan) using a C18 reversed phase column (Vydac218TP5215, 2.1×150 mm) with water:acetonitrile:formic acid(0.1%):trifluoroacetic acid (0.025%) as the mobile phase (flow: 0.2ml/min) and a gradient of 10% acetonitrile for 3 minutes, 10-20% for 3minutes, 20-25% for 21 minutes followed by a 95% wash. Peptide fragmentsin the mass range of m/z=300-2000 were detected by electrosprayionization mass spectroscopy using a LCQ ion trap, and their identitieswere confirmed by MSMS fragmentation patterns.

While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY disappeared with anapparent half time of 35 min, several intermediates were observed toaccumulate over prolonged periods (FIG. 1A). The MS intensities(m/z=300-2000 g/mol) and UV₂₈₀ absorbances of the parent peptides (SEQID NO:1) QLQPFPQPQLPY and (SEQ ID NO:3) PQPQLPYPQPQLPY were found todepend linearly on concentration in the range of 6-100 μM. The referencepeptides (SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5) QLQPFPQPQLP, (SEQ IDNO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP were generatedindividually by limited proteolysis of the parent peptides with 10 μg/mlcarboxypeptidase A (C-0261, Sigma) and/or 5.9 μg/ml leucineaminopeptidase (L-5006, Sigma) for 160 min. at 37° C. and analyzed byLC-MS as in FIG. 1.

Indeed, the subsequent processing of the peptide was substantiallyretarded (FIG. 1B). The identities of the major intermediates wereconfirmed by tandem MS, and suggested an unusually high degree ofstability of the (SEQ ID NO:8) PQPQLP sequence, a common motif in T cellstimulating peptides. Based on this data and the known amino acidpreferences of the BBM peptidases, the digestive breakdown of (SEQ IDNO:1) QLQPFPQPQLPY was reconstructed, as shown in the insert of FIG. 1B.The preferred pathway involves serial cleavage of the N-terminalglutamine and leucine residues by aminopeptidase N (APN), followed byremoval of the C-terminal tyrosine by carboxypeptidase P (CPP) andhydrolysis of the remaining N-terminal QP-dipeptide by DPP IV. As seenin FIG. 1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed by APNattack on the first two N-terminal residues) and its derivatives areincreasingly resistant to further hydrolysis. Because the high prolinecontent seemed to be a major cause for this proteolytic resistance,digestion was compared with a commercially available non-proline controlpeptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St. Louis, Mo.). Initialhydrolysis was much faster (t_(1/2)=10 min). More importantly, digestiveintermediates were only transiently observed and cleared completelywithin one hour, reflecting a continuing high specificity of the BBM forthe intermediate peptides.

Because the three major intermediate products (SEQ ID NO:6) QPFPQPQLPY,(SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP) observed during BBMmediated digestion of (SEQ ID NO:1) QLQPFPQPQLPY are substrates for DPPIV, the experiment was repeated in the presence of a 6-fold excessactivity of exogenous fungal DPP IV. Whereas the relatively rapiddecrease of the parent peptide and the intermediate levels of (SEQ IDNO:5) QLQPFPQPQLP were largely unchanged, the accumulation of DPP IVsubstrates was entirely suppressed and complete digestion was observedwithin four hours. (FIG. 1B, open bars).

To investigate the rate-limiting steps in BBM mediated digestion ofgliadin peptides from the C-terminal end, another known immunodominantpeptide derived from wheat α-gliadin, (SEQ ID NO:3) PQPQLPYPQPQLPY, wasused. Although peptides with N-terminal proline residues are unlikely toform in the small intestine (none were observed during BBM digestion of(SEQ ID NO:1) QLQPFPQPQLPY, FIG. 1A), they serve as a useful model forthe analysis of C-terminal processing since the N-terminal end of thispeptide can be considered proteolytically inaccessible due to minimalproline aminopeptidase activity in the BBM. As shown in FIG. 2, thispeptide is even more stable than (SEQ ID NO:1) QLQPFPQPQLPY. Inparticular, removal of the C-terminal tyrosine residue bycarboxypeptidase P (CPP) is the first event in its breakdown, and morethan four times slower than APN activity on (SEQ ID NO:1) QLQPFPQPQLPY(FIG. 1B). The DCP substrate (SEQ ID NO:4) PQPQLPYPQPQLP emerges as amajor intermediate following carboxypeptidase P catalysis, and is highlyresistant to further digestion, presumably due to the low level ofendogenous DCP activity naturally associated with the BBM. To confirmthe role of DCP as a rate-limiting enzyme in the C-terminal processingof immunodominant gliadin peptides, the reaction mixtures weresupplemented with rabbit lung DCP. Exogenous DCP significantly reducedthe accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after overnightincubation in a dose dependent manner (FIG. 2C). Conversely, the amountof accumulated (SEQ ID NO:4) PQPQLPYPQPQLP increased more than 2-fold inthe presence of 10 μM of captopril, a DCP-specific inhibitor, ascompared with unsupplemented BBM.

Together, the above results demonstrate that (i) immunodominant gliadinpeptides are exceptionally stable toward breakdown catalyzed by BBMpeptidases, and (ii) DPP IV and especially DCP are rate-limiting stepsin this breakdown process at the N- and C-terminal ends of the peptides,respectively. Because BBM exopeptidases are restricted to N- orC-terminal processing, it was investigated if generation of additionalfree peptide ends by pancreatic enzymes would accelerate digestion. Ofthe pancreatic proteases tested, only elastase at a high(non-physiological) concentration of 100 ng/μl was capable ofhydrolyzing (SEQ ID NO:3) PQPQLPYPQPQ^(↓)LPY. No proteolysis wasdetected with trypsin or chymotrypsin.

The above data demonstrates that proline-rich gliadin peptides areextraordinarily resistant to digestion by small intestinal endo- andexopeptidases, and therefore are likely to accumulate at highconcentrations in the intestinal cavity after a gluten rich meal. Thepathological implication of digestive resistance is strengthened by theobserved close correlation of proline content and celiac toxicity asobserved in the various common cereals (Schuppan (2000) Gastroenterology119, 234-42).

Example 2 Immunodominant Peptide of Wheat Gliadin

It has long been known that the principal toxic components of wheatgluten are a family of closely related Pro-Gln rich proteins calledgliadins. Recent reports have suggested that peptides from a shortsegment of α-gliadin appear to account for most of the gluten-specificrecognition by CD4+ T cells from Celiac Sprue patients. These peptidesare substrates of tissue transglutaminase (tTGase), the primaryauto-antigen in Celiac Sprue, and the products of this enzymaticreaction bind to the class II HLA DQ2 molecule. This exampledemonstrates, using a combination of in vitro and in vivo animal andhuman studies, that this “immunodominant” region of α-gliadin is part ofan unusually long proteolytic product generated by the digestive processthat: (a) is exceptionally resistant to further breakdown by gastric,pancreatic and intestinal brush border proteases; (b) is the highestspecificity substrate of human tissue transglutaminase (tTGase)discovered to date; (c) contains at least six overlapping copies ofepitopes known to be recognized by patient derived T cells; (d)stimulates representative T cell clones that recognize these epitopeswith sub-micromolar efficacy; and (e) has homologs in proteins from alltoxic foodgrains but no homologs in non-toxic foodgrain proteins. Inaggregate, these findings demonstrate that the onset of symptoms upongluten exposure can be traced back to a small segment of α-gliadin.Finally, it is shown that this “super-antigenic” long peptide can bedetoxified in vitro and in vivo by treatment with bacterial prolylendopeptidase, providing a strategy for peptidase therapy for CeliacSprue.

Identification of stable peptides from gastric protease, pancreaticprotease and brush border membrane peptidase catalyzed digestion ofrecombinant α2-gliadin: α2-gliadin, a representative α-gliadin(Arentz-Hansen et al. (2000) Gut 46:46), was expressed in recombinantform and purified from E. coli. The α2-gliadin gene was cloned in pET28aplasmid (Novagen) and transformed into the expression host BL21(DE3)(Novagen). The transformed cells were grown in 1-liter cultures of LBmedia containing 50 μg/ml of kanamycin at 37° C. until the OD600 0.6-1was achieved. The expression of α2-gliadin protein was induced with theaddition of 0.4 mM isopropyl β-D-thiogalactoside (Sigma), and thecultures were further incubated at 37° C. for 20 hours. The cellsexpressing the recombinant α2-gliadin were centrifuged at 3600 rpm for30 minutes. The pellet was resuspended in 15 ml of disruption buffer(200 mM sodium phosphate; 200 mM NaCl; 2.5 mM DTT; 1.5 mM benzamidine;2.5 mM EDTA; 2 mg/L pepstatin; 2 mg/L leupeptin; 30% v/v glycerol) andlysed by sonication (1 minute; output control set to 6). Aftercentrifugation at 45000 g for 45 min, the supernatant was discarded andthe pellet containing gliadin protein was resuspended in 50 ml of 7Murea in 50 mM Tris (pH=8.0). The suspension was again centrifuged at45000 g for 45 min and the supernatant was harvested for purification.

The supernatant containing α2-gliadin was incubated with 1 ml ofnickel-nitrilotriacetic acid resin (Ni-NTA; Qiagen) overnight and thenbatch-loaded on a column with 2 ml of Ni-NTA. The column was washed with7 M urea in 50 mM Tris (pH=8.0), and α2-gliadin was eluted with 200 mMimidazole, 7 M urea in 50 mM Tris (pH=4.5). The fractions containingα2-gliadin were pooled into a final concentration of 70% ethanolsolution, and two volumes of 1.5 M NaCl were added to precipitate theprotein. The solution was incubated at 4° C. overnight, and the finalprecipitate was collected by centrifugation at 45000 g for 30 min.,rinsed in water, and re-centrifuged to remove the urea. The finalpurification step of the α-2 gliadin was developed with reverse-phaseHPLC. The Ni-NTA purified protein fractions were pooled in 7 M ureabuffer and injected to a Vydac (Hesperia, Calif.) polystyrenereverse-phase column (i.d. 4.6 mm×25 cm) with the starting solvent (30%of solvent B: 1:1 HPLC-grade acetonitrile/isopropanol:0.1% TFA). SolventA was an aqueous solution with 0.1% TFA. The separation gradientextended from 30-100% of solvent B over 120 min. at a flow rate of 0.8ml/min.

TABLE 2 Amount of Peptides Digested after 15 hours 33-mer Control AControl B H1P0 <20% >90% >90% H2P0 <20% >61% >85% H3P0 <20% >87% >95%H4P0 <20% >96% >95% H5P0 <20% >96% >95%

The purity of the recombinant gliadin was >95%, which allowed for facileidentification and assignment of proteolytic products by LC-MS/MS/UV.Although many previous studies utilized pepsin/trypsin treated gliadins,it was found that, among gastric and pancreatic proteases, chymotrypsinplayed a major role in the breakdown of α2-gliadin, resulting in manysmall peptides from the C-terminal half of the protein and a few longer(>8 residues) peptides from the N-terminal half, the most noteworthybeing a relatively large fragment, the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). This peptide was ofparticular interest for two reasons: (a) whereas most other relativelystable proteolytic fragments were cleaved to smaller fragments when thereaction times were extended, the 33-mer peptide remained intact despiteprolonged exposure to proteases; and (b) three distinct patient-specificT cell epitopes identified previously are present in this peptide,namely, SEQ ID NO:59 PFPQPQLPY, SEQ ID NO:60 PQPQLPYPQ (3 copies), andSEQ ID NO:61 PYPQPQLPY (2 copies).

To establish the physiological relevance of this peptide, compositegastric/pancreatic enzymatic digestion of α2 gliadin was then examined.As expected, enzymatic digestion with pepsin (1:100 w/w ratio), trypsin(1:100), chymotrypsin (1:100), elastase (1:500) and carboxypeptidase(1:100) was quite efficient, leaving behind only a few peptides longerthan 9 residues (the minimum size for a peptide to show class II MHCmediated antigenicity) (FIG. 4). In addition to the above-mentioned33-mer, the peptide (SEQ ID NO:10) WQIPEQSR was also identified, and wasused as a control in many of the following studies. The stability of the33-mer peptide can also be appreciated, when comparing the results of asimilar experiment using myoglobin (another common dietary protein).Under similar proteolytic conditions, myoglobin is rapidly broken downinto much smaller products. No long intermediate is observed toaccumulate.

The small intestinal brush-border membrane (BBM) enzymes are known to bevital for breaking down any remaining peptides from gastric/pancreaticdigestion into amino acids, dipeptides or tripeptides for nutritionaluptake. Therefore a comprehensive analysis of gliadin metabolism alsorequired investigations into BBM processing of gliadin peptides ofreasonable length derived from gastric and pancreatic proteasetreatment. BBM fractions were prepared from rat small intestinal mucosa.The specific activities of known BBM peptidases were verified to bewithin the previously reported range. Whereas the half-life ofdisappearance of (SEQ ID NO:10) WQIPEQSR was ˜60 min. in the presence of12 ng/μl BBM protein, the half-life of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h. Therefore, thelatter peptide remains intact throughout the digestive process in thestomach and upper small intestine, and is poised to act as a potentialantigen for T cell proliferation and intestinal toxicity in geneticallysusceptible individuals.

Verification of proteolytic resistance of the 33-mer gliadin peptidewith brush border membrane preparations from human intestinal biopsies:To validate the above conclusions, derived from studies with rat BBMpreparations, in the context of human intestinal digestion, BBMpreparations were prepared from a panel of adult human volunteers, oneof whom was a Celiac Sprue patient in remission, while the rest werefound to have normal intestinal histology. (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, (SEQ ID NO:1) QLQPFPQPQLPY (aninternal sequence from the 33-mer used as a control), (SEQ ID NO:10)WQIPEQSR and other control peptides (100 μM) were incubated with BBMprepared from each human biopsy (final aminopeptidase N activity of 13μU/μl) at 37° C. for varying time periods. While (SEQ ID NO:1)QLQPFPQPQLPY, (SEQ ID NO:10) WQIPEQSR and other control peptides werecompletely proteolyzed within 1-5 h, the long peptide remained largelyintact for at 19 hours. These results confirm the equivalence betweenthe rat and human BBM for the purpose of this study.

Verification of proteolytic resistance of the 33-mer gliadin peptide inintact animals: The proteolytic resistance of the 33-mer gliadinpeptide, observed in vitro using BBM from rats and humans, was confirmedin vivo using a perfusion protocol in intact adult rats (Smithson andGray (1977) J. Clin. Invest. 60:665). Purified peptide solutions wereperfused through a 15-20 cm segment of jejunum in a sedated rat with aresidence time of 20 min., and the products were collected and subjectedto LC-MS analysis. Whereas >90% of (SEQ ID NO:1) QLQPFPQPQLPY wasproteolyzed in the perfusion experiment, most of the 33-mer gliadinpeptide remained intact. These results demonstrate that the 33-merpeptide is very stable as it is transported through the mammalian uppersmall intestine.

The 33-mer gliadin peptide is an excellent substrate for tTGase, and theresulting product is a highly potent activator of patient-derived Tcells. A number of recent studies have demonstrated that regiospecificdeamidation of immunogenic gliadin peptides by tTGase increases theiraffinity for HLA-DQ2 as well as the potency with which they activatepatient-derived gluten-specific T cells. It has been shown thespecificity of tTGase for certain short antigenic peptides derived fromgliadin is higher than its specificity toward its physiological targetsite in fibronectin, for example, the specificity of tTGase for theα-gliadin derived peptide (SEQ ID NO: 3) PQPQLPYPQPQLPY is 5-fold higherthan that for its target peptide sequence in fibrinogen, its naturalsubstrate. The kinetics and regiospecificity of deamidation of the33-mer α-gliadin peptide identified as above were therefore measured.The k_(cat)/K_(M) was higher than that reported for any peptide studiedthus far: k_(cat)/K_(M)=440 min−1 mM−1 for (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min−1 mM−1 forPQPQLPY and kcat/KM=350 min−1 mM−1 for (SEQ ID NO: 3) PQPQLPYPQPQLPY.

Moreover, LC-MS-MS analysis revealed that (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated by tTGaseat the underlined residues. Since tTGase activity is associated with thebrush border membrane of intestinal enterocytes, it is likely thatdietary uptake of even small quantities of wheat gluten will lead to thebuild-up of sufficient quantities of this 33-mer gliadin peptide in theintestinal lumen so as to be recognized and processed by tTGase.

Structural characteristics of the 33-mer gliadin peptide and itsnaturally occurring homologs: Sequence alignment searches using BLASTPin all non-redundant protein databases revealed several homologs(E-value <0.001) of the 33-mer gliadin peptide. Interestingly, foodgrainderived homologs were only found in gliadins (from wheat), hordeins(from barley) and secalins (from rye), all of which have been proven tobe toxic to Celiac patients. See FIG. 6. Nontoxic foodgrain proteins,such as avenins (in oats), rice and maize, do not contain homologoussequences to the 33-mer gliadin. In contrast, a BLASTP search with theentire α2-gliadin sequence identified foodgrain protein homologs fromboth toxic and nontoxic proteins. Based on available informationregarding the substrate specificities of gastric, pancreatic and BBMproteases and peptidases, it is predicted that, although most glutenhomologs to the 33-mer gliadin peptide contained multiple proteolyticsites and are therefore unlikely to be completely stable towarddigestion, several sequences from wheat, rye and barley are expected tobe comparably resistant to gastric and intestinal proteolysis. Thestable peptide homologs to the 33-mer σ2-gliadin peptide are (SEQ IDNO:13) QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- and α6-gliadins);(SEQ ID NO:14) QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein);(SEQ ID NO: 15) QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin); (SEQ IDNO:16) QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from ω-secalin). These stablepeptides are all located at the N-terminal region of the correspondingproteins. The presence of proline residues after otherwise cleavableresidues in these peptides would contribute to their proteolyticstability.

The unique primary sequence of the 33-mer gliadin peptide also hadhomologs among a few non-gluten proteins. Among the strongest homologswere internal sequences from pertactin (a highly immunogenic proteinfrom Bordetella pertussis) and a mammalian inositol-polyphosphate5-phosphatase of unknown function. In both cases available informationsuggested that the homology could have biologically relevance. Forexample, the region of pertactin that is homologous to the 33-mergliadin peptide is known to be part of the immunodominant segment of theprotein. In the case of the homologous phosphatase, the correspondingpeptide region of the phosphatase is known to be responsible forvesicular trafficking of the phosphatase to the cytoplasmic Golgi. Inanalogy with the current picture of how gliadin peptides are presentedto HLA-DQ2 via a tTGase mediated pathway, these Pro-Gln-rich segments ofboth pertactin and the phosphatase are likely to be good tTGasesubstrates. To test this hypothesis, the corresponding peptides weresynthesized, and the selectivity of tTGase for these peptides wasmeasured. As predicted, both peptides were found to be good substratesof tTGase. The tTGase enzyme plays a central role in receptor mediatedendocytosis of several biologically important proteins. The biologicalactivities of both pertactin and the phosphatase may depend on tTGasemediated trafficking.

Secondary structural studies using circular dichroism spectroscopy onthe 33-mer gliadin peptide as well as its homologs from pertactin andthe inositol-polyphosphate 5-phosphatase demonstrate that these peptideshave strong type II polyproline helical character. In addition toreinforcing the proteolytic resistance of these peptides, the type IIpolyproline helical conformation is also likely to enhance theiraffinity for class II MHC proteins.

Although gluten proteins from foodgrains such as wheat, rye and barleyare central components of a nutritious diet, they can be extremely toxicfor patients suffering from Celiac Sprue. To elucidate the structuralbasis of gluten toxicity in Celiac Sprue, comprehensive proteolyticanalysis was performed on a representative recombinant gliadin underphysiologically relevant conditions. An unusually long andproteolytically stable peptide product was discovered, whosephysiological relevance was confirmed by studies involving brush bordermembrane proteins from rat and human intestines as well as intestinalperfusion assays in live rats. In aggregate, these data demonstrate thatthis peptide and its homologs found in other wheat, rye and barleyproteins are the “root cause” of the initial inflammatory response todietary wheat in Celiac Sprue patients in remission.

Example 3

Human leukocyte antigen DQ2 is a class II major histocompatibilitycomplex protein that plays a critical role in the pathogenesis of CeliacSprue by binding to epitopes derived from dietary gluten and triggeringthe inflammatory response of disease-specific T cells. Inhibition of DQ2mediated antigen presentation in the small intestinal mucosa of CeliacSprue patients therefore represents a potentially attractive mode oftherapy for this widespread but unmet medical need. Starting from apro-inflammatory, proteolytically resistant, 33-residue peptide, (SEQ IDNO:12) LQLQPFPQPEL PYPQPELPYPQPELPYPQPQPF, we embarked upon a systematiceffort to dissect the relationships between peptide structure and DQ2affinity, and to translate these insights into prototypical DQ2 blockingagents. Three structural determinants within the first 20 residues ofthis 33-mer peptide, including a (SEQ ID NO: 18) PQPELPYPQ epitope, itsN-terminal flanking sequence and a downstream Glu residue, were found tobe critical for DQ2 recognition. Guided by the X-ray crystal structureof DQ2, the L11 and L18 residues in the truncated 20-mer analogue werereplaced with sterically bulky groups so as to retain high DQ2 affinitybut abrogate T cell recognition. A dimeric ligand synthesized byregiospecific coupling of the 20-mer peptide with a bifunctional linker,was identified as an especially potent DQ2 binding agent. Two suchligands were able to attenuate the proliferation of disease-specific Tcell lines in response to gluten antigens, and therefore representprototypical examples of pharmacologically suitable DQ2 blocking agentsfor the potential treatment of Celiac Sprue.

Inhibition of antigen presentation by blocking a disease-specific MHC onantigen presenting cells with peptide (and occasionally non-peptide)ligands has been previously explored as a therapeutic strategy forautoimmune diseases such as multiple sclerosis, rheumatoid arthritis,diabetes, and experimental autoimmune encephalomyelitis. Such therapy isof particular interest for the treatment of Celiac Sprue. First, to dateno non-dietary treatment has been developed for this widespread,lifelong disease; as such, there is an acute unmet need. Second, incontrast to the organs affected by most other autoimmune diseases, thesmall intestine is readily accessible via oral administration of atherapeutic candidate. Finally, and perhaps most importantly, among HLAmediated diseases, Celiac Sprue is unique in that an environmentaltrigger (dietary gluten) has been identified and extensively dissectedat an immunological level. In turn, these studies have led to theidentification of proteolytically resistant gluten peptides that aregenerated by physiological processes and are efficiently presented todisease associated T cells in a DQ2 restricted fashion. Thus, if thesenaturally occurring T cell stimulatory agents can be transformed intoinhibitors of DQ2 mediated antigen presentation, they can be consideredas appropriate medicinal leads for Celiac Sprue.

A Pro- and Gln-rich 33-mer peptide from α2-gliadin, (SEQ ID NO:12)LQLQPFPQPELPYPQPE LPYPQPELPYPQPQPF (transglutaminase-catalyzed Gln->Gluchanges underlined), is a particularly interesting lead peptide for thispurpose. Its extreme resistance to breakdown by luminal proteases andintestinal brush-border enzymes allows it to persist for a considerableduration in the upper small intestine, the primary affected region ofthe gastrointestinal tract in a Celiac Sprue patient. Not only does thispeptide have a high affinity for HLA-DQ2, it is displayed on the surfaceof antigen presenting cells with unusual robustness. Not surprisingly,it is a potent proliferative trigger of gluten-responsive T cells fromsmall intestinal biopsy samples of all DQ2 Celiac Sprue patients testedthus far. Although this peptide is multivalent (it has 6 overlappingcopies of 3 epitopes), it binds to HLA-DQ2 with a 1:1 stoichiometry. Ithas a considerably higher affinity for DQ2 than any of its constituentepitopes (SEQ ID NO:17) PFPQPELPY, (SEQ ID NO:18) PQPELPYPQ and (SEQ IDNO:58) PYPQPELPY. Together, these observations led us to hypothesizethat the 33-mer peptide harbors secondary interactions with DQ2 outsidethe core antigen binding pocket. Understanding the precise nature ofthese interactions would therefore be a critical prerequisite forexploiting its potential as a medicinal lead in the design of DQ2blocking agents.

In this report we have dissected the structural determinants of thehigh-affinity interaction between the 33-mer peptide and HLA-DQ2. Basedon these findings, we designed and synthesized simple analogues of the33-mer peptide that retain its strong affinity for DQ2 but are notrecognized by 33-mer responsive T cells from Celiac biopsies. Theability of these putative blocking agents to inhibit T cellproliferation in response to gluten antigens was also demonstrated.These peptides represent the first prototypical examples ofpharmacologically relevant DQ2 blocking agents for potential treatmentof Celiac Sprue.

Experimental Section

DQ2 expression and purification. Soluble DQ2 molecules were expressedand purified as previously described. Briefly, the soluble extracellulardomains of the DQ2 α and β chains were co-expressed in High Five insectcells using a pAcAB3 baculovirus expression system, and wereaffinity-purified using the anti-DQ2 mAb 2.12.E11. The sequence (SEQ IDNO:55) QLQPFPQPELPYwas fused to the N-terminus of the DQ2 β-chain by a15-residue linker (SEQ ID NO:56) (GAGSLVPRGSGGGGS), which includes athrombin site. A complementary Fos/Jun leucine zipper pair wasengineered at the C-terminal ends of α and β chains, respectively, withintervening factor Xa proteolysis sites, to increase the heterodimerstability during protein expression.

The concentration of HLA-DQ2 was determined by UV spectrophotometry at280 nm using the absorption coefficient factor 75,700 cm⁻¹M⁻¹ ascalculated from the contents of tyrosine, tryptophan and cystine in theDQ2 sequence (22). Prior to use in binding experiments with exogenousligands, the DQ2-ligand fusion protein was first treated with ˜2% w/wthrombin in pH 7.3 PBS at 0° C. for 2 h.

Peptide synthesis, labeling and purification. All peptides used in thisstudy were synthesized using Boc/HBTU chemistry starting fromN-α-t-Boc-L-aminoacyl-phenylacetamidomethyl (PAM) resin. Peptides werelabeled at their N-termini while still attached to the resin with 5-(and6-) carboxyfluorescein, 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimidehydrochloride (EDC.HCl), and 1-Hydroxy-7-azabenzotriazole (HOAt) in1:1:1 ratio in dimethylformamide as the solvent. Following cleavage ofthe peptidyl resin in trifluoroacetic acid/trifluoromethanesulfonicacid/thioanisole (TFA/TFMSA/thioanisole 10:1:1, v/v/v) for 4 h, thecrude peptides were precipitated in cold ether and dissolved in 1:1 v/vacetonitrile/water. The peptides were purified by reverse-phase HPLC ona semi-preparative C₁₈ column using a water-acetonitrile gradient in0.1% (v/v) TFA. The identity and purity of the peptides were confirmedby electrospray mass spectrometry and analytical reverse-phase HPLC. Thepeptides were lyophilized and stored at −20° C. Prior to use, peptidestock solutions were prepared in 10 mM PBS with 0.02% sodium azide, andtheir concentrations were determined by UV-Vis spectrophotometry at 495nm using the absorption coefficient factor 80,200 cm⁻¹M⁻¹ for thefluorescein-labeled peptide. The integrity of the peptide stocks wasmonitored by analytical HPLC every several months.

Peptide derivatives 17-22 (see FIG. 10) were synthesized withN-α-t-Boc-N-ε-Fmoc-L-lysine. The Fmoc-protected side chain of lysine wasdeprotected after synthesis of the full-length peptide by washing theresin twice in 20% piperidine in dimethylformamide for 15 min. Then 1 gof succinimide anhydride dissolved in 2 ml dimethylformamide was addedto the resin for 30 min. The extent of amide formation was monitored bythe ninhydrin test.

The dimeric peptide 22 was synthesized from pure monomeric fluoresceinlabeled peptide 17. Fluorescein-conjugated 17 was mixed with bis-d PEG 6NHS ester (Quanta Biodesign) in 2:1 ratio in either dimethylformamidewith 10% v/v diisopropylethylamine as base, or in pH 9 phosphatesolution. The reaction was monitored by analytical reverse phase HPLCusing a C₁₈ column. The product peak eluted 2 min after the monomericstarting material, and was purified by preparative reverse phase HPLC.Mass spectrometric analysis revealed that it had the expected molecularweight of 5858.8 (22+Na⁺, exp. MW 5858). The concentration of thisfluorescent dimeric peptide was quantified by using the absorptioncoefficient factor 160,400 cm⁻¹M⁻¹ at 495 nm.

Peptide exchange assay. For peptide exchange experiments, the DQ2heterodimer purified as described above was incubated withfluorescein-conjugated ligands in a 25:1 ratio (i.e. 4.7 μM DQ2 with0.18 μM fluorescent peptide). Incubations were performed at 37° C. in a1:1 mixture of PBS buffer (10 mM Pi, 150 mM NaCl, pH 7.3, supplementedwith 0.02% NaN₃) and McIlvaine's citrate-phosphate buffers (pH 5 or pH7) such that the final pH was either 5.5 or 7.3, respectively. Peptidebinding was measured by high performance size exclusion chromatography(HPSEC) (17). 1 μl of reaction mixture was diluted into 14 μl PBS. 12.5μl of the diluted material was injected onto a BioSeptember 3000 sizeexclusion column (Phenomenex), and eluted with PBS buffer at 1 ml/min.The DQ2-peptide complex eluted at 8.5 min, with free peptides emerging˜2 min later. The fluorescence signal was recorded using an in-lineShimadzu RA35 fluorescent detector with excitation wavelength set at 495nm and emission detection set at 520 nm. Peak areas corresponding to theDQ2-peptide complex and the free peptide were used to calculate thefractional yield of the DQ2-fluoresceinated peptide complex.

Peptide dissociation assay. For dissociation experiments,DQ2-fluoresceinated peptide complexes were prepared by incubatingthrombin treated DQ2 (3-5 μM) with 20-fold excess fluorescein-conjugatedpeptides at pH 5.5 for 25 hours. The buffer composition was a 1:1mixture of 10 mM PBS buffer and pH 5.1 McIlvaine's citrate-phosphatebuffer (24), such that the final pH was 5.5. Excess free peptide wasseparated from the complex on a chilled spin column (Bio-Rad) packedwith Sephadex G50 superfine medium and blocked with 1% BSA solution tominimize the binding of DQ2 to the column. Spin columns were pre-washedwith pH 7.3 PBS buffer, and the fluorescein-conjugated peptide+DQ2mixture was applied to the column. The DQ2-fluoresceinated peptidecomplex was eluted in a volume of ˜230 μl in pH 7.3 PBS buffer.Typically, this DQ2-peptide fraction contained <10% of free peptide. Thesolution was immediately adjusted to pH 5.5 or pH 7.3, and 20 μM of atight DQ2 binding peptide (SEQ ID NO:57) (AAIAAVKEEAF) was added toprevent the re-binding of dissociated fluorescent peptide to DQ2.Kinetic measurements of ligand dissociation were performed at 37° C.,and a time course was obtained by injecting 20 μl aliquots into HPSECcolumn.

T cell proliferation assays. The DQ2 homozygous B-lymphoma cell line(LCL) VAVY cells were irradiated with 12,000 rads of γ-irradiation orfixed with 1% paraformaldehyde for 10 minutes as indicated, andincubated with the appropriate peptides overnight in media containing10% fetal bovine serum, 2% human serum, penicillin and streptomycin at acell density of 2×10⁶ cells/ml in 96-well plates. The next day, thevolume was doubled to yield a cell density of 1×10⁶ cells/ml, 50 μl ofwhich was placed into a U-bottom 96-well plate. An equal volume ofT-cells (50 μl of 1×10⁶ cells/ml) was added to each well, and cells wereincubated at 37° C. and 5% CO₂ for 48 h, at which point 0.5 μCi/well of[methyl-³H] thymidine (Amersham, TRK120) was added. Cells were incubatedfor an additional 12-14 h and then frozen. After thawing, incorporatedthymidine was collected on a filter mat (Wallac, 1205-401) using aTomtec cell harvester, and counted using a Wallac 1205 Betaplate liquidscintillation counter.

Results

SAR analysis of the binding of the 33-mer peptide to HLA-DQ2. Earlierstudies have demonstrated that the highly immunogenic 33-mer peptidefrom α2-gliadin, (SEQ ID NO:12) LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF,potently displaces pre-bound ligands from the DQ2 binding pocket andalso has a long dissociation half-life. These features, together withits natural proteolytic resistance, make this 33-mer peptide anattractive target for engineering an HLA-DQ2 blocking agent. To dissectits structure-activity relationships (SAR), several analogues weresynthesized and initially evaluated in peptide exchange assays over aperiod of 45 h (FIG. 1). Although the percentage of bound peptide isonly reported at the 45 h end-point (i.e. at equilibrium), thehalf-maximum binding times for several of these peptides were obtained,and found to be similar. Therefore, the equilibrium occupancy isrepresentative of DQ2 avidity.

Several noteworthy observations emerge from the data summarized in FIG.8. First, consistent with earlier data, it is evident that the αIIepitope (SEQ ID NO:18) (PQPELPYPQ) is the primary binding site in the33-mer peptide 1. For example, the minimal αII epitope bearing peptide3, has a binding maximum of 7% at pH 5.5, whereas the minimal αI epitopebearing peptide 2 saturates at less than 1% at pH 5.5 and isundetectable at pH 7.3. Second, comparison of the DQ2 binding propertiesof the minimal αII epitope 3 with longer analogues (e.g. 6 and 7)reveals that binding efficiency increases as a greater proportion of the33-mer sequence is included at the C-terminal side of the epitope.Similarly, comparison of the binding properties of the minimal peptide 3with the N-terminally elongated peptide 4 suggests that the N-terminusof the 33-mer contributes significantly to its DQ2 binding potency.Together, these findings argue that structural determinants on bothsides of the core αII epitope sequence of the 33-mer influence itsimmunological characteristics. The importance of flanking sequences isalso reinforced when one compares 21-mer peptide 7 with analogues thatare incrementally extended on both sides (8, 9, 10 and 1).Interestingly, the difference between peptides 7 and 8 is morepronounced at pH 7 than at pH 5.

The 33-mer peptide 1 contains three Glu residues corresponding to threetransglutaminase-catalyzed deamidation sites. To investigate the role ofmultiple deamidation sites, we synthesized and tested all possibledeamidation analogues of the 33-mer (peptides 11-16). Q10E (11) hashigher affinity for DQ2 than either Q17E (12) or Q24E (13) at both pH 5and pH 7, arguing that DQ2 binding of the 33-mer is primarily centeredat the N-terminal αII epitope, although the other αII epitopes can alsobind effectively in the MHC pocket. Notably however, introduction of asecond deamidation site (e.g. Q10EQ17E (14) or Q10EQ24E (15))significantly enhances affinity relative to Q10E (11), suggesting that asecond acidic residue C-terminal to the core αII epitope is importantfor the optimal immunological properties of the 33-mer. This is alsosupported by the observation that the doubly deamidated analogueQ17EQ24E (16) approximates the 33-mer binding characteristics.

Taken together, the above data support that a peptide comprising: (i)the first αII epitope of the 33-mer, (ii) the N-terminal sequence, and(iii) adequate C-terminal sequence to include at least one additionaldeamidation site, would be the shortest 33-mer analogue that couldcapture the essential DQ2 binding characteristics of the naturalproduct. To test this hypothesis, we synthesized the 20-mer peptide 5,and quantitatively compared its ability to bind DQ2 in peptide exchangeand dissociation assays. The results, summarized in FIG. 8, show thatpeptide 5 is comparable to the 33-mer peptide 1 in all in vitro assays.The immunological relevance of these biochemical findings was verifiedin T cell proliferation assays using a polyclonal T-cell line thatresponds to all three a epitopes equally well and a clonal T-cell linethat responds to the all epitope exclusively. As shown in FIG. 9A, the Tcell stimulatory characteristics of peptides 1 and 5 are very similar,both of which are substantially more potent T cell antigens than theminimal αII epitope peptide 3. To verify that the enhanced T cellantigenicity of peptides 1 and 5 is due to superior DQ2 binding affinityrather than the ability of these peptides to stimulate a largerpopulation of α-epitope responsive T cells, the experiment was repeatedusing a clonal T cell line specific for the αII epitope. FIG. 2B showsthat peptides 1 and 5 again have a comparable T cell stimulatorycapacity that exceeds that of peptide 3. Thus, in view of itsconsiderably shorter length, peptide 5 serves as a lead for the designof HLA-DQ2 blocking ligands for treatment of Celiac Sprue.

Design and in vitro evaluation of DQ2 blocking peptides. A major goal ofthis research is to design and characterize medicinally appropriateligands that form tight, long-lived complexes with HLA-DQ2 on thesurface of antigen presenting cells in the Celiac small intestine, butare not recognized by disease specific T cells. Toward this end wesynthesized analogues of the 20-mer peptide 5, and evaluated their DQ2binding properties. Our initial design targeted L11 and L18 residues ofpeptide 5 as modification sites. L11 was chosen based on the crystalstructure of the αI-DQ2 complex, which suggested that the residue at thecorresponding position (i.e. the P5 Pro residue) points away from theDQ2 protein surface. Consistent with this observation, epitope scanningexperiments have also shown that the antigen binding pocket of DQ2 canaccommodate a spectrum of amino acids at the P5 position. Therefore, theL11K analogue (17) shown in FIG. 10 was synthesized. In addition, sincepeptide 5 can bind to HLA-DQ2 in the αI (SEQ ID NO:17) (PFPQPELPY) andαII (SEQ ID NO:18) (PQPELPYPQ) epitope registers, we also wished tointroduce modifications in the downstream αIII epitope. For this reasonL18K (18) was also synthesized. (Like the P5 residue, the P7 residuealso points away from the DQ2 protein surface.)

Surprisingly, both 17 and 18 bound poorly to DQ2 at either pH 5 or pH 7(FIGS. 11A and 11B). We speculated that this poor affinity may be due toformation of a salt bridge between the Lys residue at positions 11 or 18and the critical Glu residue at the preceding position. To test thishypothesis, the Lys side chains in both compounds were extended with asuccinyl group, yielding peptides 19 and 20 (FIG. 3) with a negativecharge at residues 11 and 18, respectively. As shown in FIGS. 11A and11B, peptides 19 and 20 bound to DQ2 with considerably higher affinity.We therefore also synthesized peptide 21, which possesses the abovechanges at both positions 11 and 18. Peptide 21 bound comparably wellwith DQ2 as peptide 5, both in exchange experiments (FIG. 4C) anddissociation experiments.

A number of independent investigations have highlighted the feasibilityof greatly enhancing ligand avidity to a biological target by theengineering of multivalent ligands. Therefore, in an attempt to furtherimprove DQ2 affinity, a dimeric ligand (22) was synthesized bycrosslinking monomeric L11K via a hexa-ethylene glycol (6-PEG)bifunctional linker through the Lys side chains (FIG. 10). Remarkably,this ligand has considerably improved binding affinity for DQ2 ascompared to all gluten peptides evaluated thus far, including the 33-merpeptide 1 (FIG. 12).

Since the modified side-chains of ligands 19-22 are oriented toward theT cell face of the DQ2-peptide complex, we anticipated that thesemodifications were likely to alter the T cell recognition properties ofthese peptides. As an initial test of this hypothesis, peptides 5 and19-21 were labeled with biotin at their N termini and mixed with liveVAVY (DQ2 homozygous) antigen presenting cells. The extent to whichthese peptides were presented on the cell surface was visualized usingfluorescent streptavidin. After 24 h, the intensity of staining of theVAVY cell surface was found to be comparable by confocal microscopy inthe presence of all labeled peptides.

Next, we wished to investigate the extent to which compounds 19-22 wereable to elicit a proliferative response from gluten responsivepolyclonal T cell lines derived from small intestinal biopsies of CeliacSprue patients. For this experiment, a T cell line that is stronglyresponsive to the 33-mer peptide 1 (or alternately peptide 5) was used.As shown in FIG. 13, peptide 20 elicited the strongest T cell response,presumably because it contained an unmodified all (SEQ ID NO:18)(PQPELPYPQ) epitope. A low but measurable T cell response was alsoobserved for peptides 19 and 22, perhaps because they contained anunmodified αIII (SEQ ID NO:58) (PYPQPELPY) epitope. In contrast, thedoubly modified peptide 21, which lacks any of the intact α-gliadinepitopes, completely abrogates T cell recognition. Together, theseresults suggest that compounds 21 and 22 can be presented on the surfaceof DQ2 antigen presenting cells, but are relatively unrecognized bygluten specific T cells found in the small intestines of Celiac Spruepatients. Given their intrinsic proteolytic stability, these peptideswere therefore evaluated as prototypical DQ2 blocking agents.

To assess the DQ2 blocking properties of the most promising compounds,21 and 22, paraformaldehyde-fixed VAVY cells were incubated for 12 hourswith varying concentrations of antigenic peptide 5 in the presence orabsence of 5 μM blocker peptide, and the resulting antigen presentingcells were then mixed with gluten responsive T cells under appropriateculture conditions (FIG. 14). A significant increase in EC₅₀ wasobserved in the presence of compounds 21 and 22, indicating that thesesynthetic peptides were indeed capable of competitively blocking T cellproliferation by potent gluten antigens such as peptide 5.

Discussion

Several recent reports have highlighted the exceptional pathogeniccharacteristics of a 33-mer peptide from α2 gliadin, (SEQ ID NO:12)LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, using disease specific T cellsderived from small intestinal biopsies of Celiac Sprue patients. Thegoal of this research is to evaluate the feasibility of transformingthis potent pro-inflammatory natural product into a fundamentally newtype of anti-inflammatory agent for potential use in Celiac Spruepatients. To this end we have dissected the structure-functionrelationships of the 33-mer peptide, and have harnessed these insights,together with our recent X-ray crystal structure of HLA-DQ2, to designprototypical DQ2 blocking ligands.

The 33-mer peptide is a multivalent antigen possessing at least sixoverlapping copies of three distinct epitopes (designated earlier as αI,αII and αIII epitopes, corresponding to the sequences (SEQ ID NO:17)PFPQPELPY, (SEQ ID NO:18) PQPELPYPQ and (SEQ ID NO:58) PYPQPELPY,respectively). However, its predominant mode of DQ2 interaction involvesformation of stable monomeric complexes (1:1 stoichiometry). Detailedequilibrium binding and kinetic analysis of the 33-mer peptide toHLA-DQ2 led us to hypothesize that this peptide derives its DQ2 aviditythrough a combination of interactions between one or more core epitopesas well as flanking sequences. Therefore, a first objective of thisstudy was to identify the preferred binding epitope(s), the structuraldeterminants in the flanking sequences, and the precise contributions ofthe three Glu residues, each of which is generated by post-translationaldeamidation of a naturally occurring Gln residue by the humantransglutaminase 2 enzyme.

We synthesized truncated analogues (peptides 2, 3, 6-10) as well assite-directed variants (peptides 11-16) of the 33-mer peptide 1, andtested their ability to displace a pre-existing ligand in the bindingpocket of a purified, soluble form of HLA-DQ2 (FIG. 8). Our findingssuggest that the αII epitope centered at E10 is the most preferredregister for 33-mer binding to DQ2. This is presumably due to the higheravidity of the αII epitope for DQ2 as compared to αI and αIII epitopes,as well as the fact that the first αII epitope in the 33-mer peptide canuniquely leverage secondary interactions between the N-terminal (SEQ IDNO:32) LQLQPF sequence and a yet to be determined binding site onHLA-DQ2. Additionally, a second deamidation site located in theC-terminal sequence also facilitates DQ2 binding of this αII epitope.Thus, the 20-mer peptide 5 binds to DQ2 equally well as the 33-merpeptide 1, whereas peptide 6 (which lacks the N-terminal flank) has aconsiderably lower affinity for DQ2 than peptide 5 (which has thisflanking sequence). The good correlation between DQ2 binding and T cellproliferative capacity was also verified by showing that peptide 5 hascomparable T cell antigenicity as 1 (FIG. 9).

The X-ray crystal structure of HLA-DQ2 bound to the αI gliadin epitopehad revealed that some amino acid side chains from the epitope aredeeply buried in the DQ2 binding pocket, whereas others face outward,presumably in the direction of the T cell receptor. L11 and L18 inpeptide 5 are examples of the latter category of residues, regardless ofwhether they are recognized by DQ2 as part of an αII epitope (where theybind in the P5 binding pocket) or the αI or αIII epitopes (where theybind in the P7 pocket). Leu->Lys substitutions at these positionstherefore provided functional handles for further modification. Severalanalogues (17-21) of peptide 5 were evaluated; of these, compounds 19,20, and 21 had similar affinity for HLA-DQ2 as unsubstituted peptide 5(FIG. 11). In T cell assays, whereas peptides 19 and 20 did retain someability to stimulate the proliferation of gluten specific T cells fromintestinal biopsies of Celiac Sprue patients, the doubly modifiedpeptide 21 is unable to stimulate an αI, αII, and αIII epitoperesponsive T cell line (FIG. 13). In the presence of 5 μM 21, the EC₅₀of antigenic peptide 5 against gluten specific T cells is increased.

An alternative design of potential DQ2 blocking agents involveddimerization of peptide 17 through the Lys-11 side chain. This strategyhas borne fruit in the study of protein-protein interactions in otherbiological contexts. Our prototypical dimeric peptide 22 with ahexa-ethylene glycol bifunctional linker had a substantially enhancedaffinity for HLA-DQ2 than both peptides 1 and 5 at pH 5 or pH 7.Moreover, compound 22 showed remarkably enhanced binding kinetics,reaching half maximal DQ2 occupancy at 2.5 hr instead of ˜8 hr for the33-mer peptide 1 (FIG. 5). This rapid binding capacity could be apharmacologically useful property in the context of inhibiting antigenpresentation. In support of this anti-inflammatory potential, additionof 5 μM compound 22 also resulted in a significant elevation of the EC₅₀of antigenic peptide 5.

In summary, our studies reported here have led to the development of animproved insight into the structure-activity relationships of the highlyimmunogenic 33-mer peptide from α2-gliadin, and in turn, to the designof peptidic analogues of this peptide that bind tightly to HLA-DQ2 butare not recognized by Celiac Sprue associated T cells. The design offuture generations of such DQ2 blocking agents will require in-depthbiological evaluations of these promising synthetic agents.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A purified oligopeptide of from about 14 amino acids in length toabout 33 amino acids in length, comprising the epitope sequence (SEQ IDNO:43) PQPEKPYPQ.
 2. The purified oligopeptide of claim 1, wherein saidpeptide comprises as the amino terminal sequence, SEQ ID NO:32, LQLQPF.3. The purified oligopeptide of claim 1, wherein the carboxy terminalsequence is SEQ ID NO:44 PELPY, or SEQ ID NO:45 PEKPY.
 4. The purifiedoligopeptide of claim 1, wherein one or more lysine residues areconjugated to a group that provides for steric hindrance of interactionsbetween the peptide and a cognate receptor.
 5. The purified oligopeptideof claim 4, wherein said lysine residue is conjugated to succinic acid;benzyloxycarbonyl group; t-Butoxycarbonyl group;9-fluorenylmethoxycarbonyl group; phthalimides; or polyethylene glycol.6. A pharmaceutical composition comprising the oligopeptides of claim 1,and a pharmaceutically acceptable excipient.
 7. A method for diagnosingCeliac Sprue in an individual, said method comprising determiningwhether an oligopeptide of claim 1 stimulates agglutination ofanti-gliadin antibodies, anti-tTGase antibodies, or combinations thereoffrom said individual, and correlating an ability to stimulateagglutination with a positive diagnosis of Celiac Sprue.
 8. The methodof claim 7, wherein said tissue is a mucosal tissue selected from thegroup consisting of oral, nasal, lung, and intestinal mucosal tissue. 9.The method of claim 7, wherein said bodily fluid is selected from thegroup consisting of blood, sputum, urine, phlegm, lymph, and tears. 10.The method of claim 7, wherein said individual has not consumed glutenfor an extended period of time.
 11. The method of claim 10, wherein saidextended period of time is selected from the group consisting of oneday, one week, one month, and one year prior to the performance of thediagnostic method.
 12. The method of claim 7, wherein said individualhas not had an endoscopy.
 13. The method of claim 7, wherein saidindividual is the subject of a therapy intended to treat Celiac Sprue oris in a clinical trial conducted to evaluate such a therapy.
 14. AnHLA-binding peptide inhibitor; wherein said inhibitor is an analog of animmunogenic gluten oligopeptide of at least about 8 residues in length,wherein the immunogenic gluten oligopeptide is altered by thereplacement of one or more amino acids; and wherein said analog bindstightly to HLA molecules; is proteolytically stable; and does notactivate disease-specific T cells.
 15. The HLA-binding peptide inhibitorof claim 14, wherein said analog comprises one or more naturallyoccurring amino acids, non-naturally occurring amino acids, modifiedamino acids, or amino acid mimetics.
 16. The HLA-binding peptideinhibitor of claim 15, wherein said analog is further derivatized toreduce the affinity of the analog for disease-specific T cell receptors.17. The HLA-binding peptide inhibitor of claim 16, wherein saidimmunogenic gluten oligopeptides comprises at least one PXP motif. 18.The HLA-binding peptide inhibitor of claim 14, wherein said immunogenicgluten oligopeptides comprises a sequence selected from the groupconsisting of: (SEQ ID NO: 36) PQPELPY; PFPQPELPYP, (SEQ ID NO: 47)PQPELPYPQPQLP, (SEQ ID NO: 48) PQQSFPEQQPP, (SEQ ID NO: 49)VQGQGIIQPEQPAQ, (SEQ ID NO: 50) FPEQPQQPYPQQP, (SEQ ID NO: 51)FPQQPEQPYPQQP, FSQPEQEFPQPQ; PFPQPQLPY, PQPQLPYPQ, (SEQ ID NO: 17)PFPQPELPY; (SEQ ID NO: 58) PYPQPELPY and PYPQPQLPY.
 19. The HLA-bindingpeptide inhibitor of claim 14, wherein said inhibitor comprises thesequence PXPQPELPY, where X is Tyr, Trp, Arg, Lys, p-iodo-Phe,3-iodo-Tyr, p-amino-Phe, 3-amino-Tyr, hydroxylysine, ornithine, Asp orGlu.
 20. The HLA-binding peptide inhibitor of claim 19, wherein saidinhibitor is further derivatized to reduce the affinity of the analogfor disease-specific T cell receptors.
 21. The HLA-binding peptideinhibitor of claim 14, wherein said inhibitor is further modified toincrease binding potency to an MHC molecule.
 22. The HLA-binding peptideinhibitor of claim 14, wherein said inhibitor comprises the sequencePFPQX₁ELX₂Y, where X₁ and X₂ are independently selected from4-hydroxy-Pro, 4-amino-Pro, or 3-hydroxy-Pro, and proline, with theproviso that at least one of X₁ and X₂ is a residue other than proline.23. The HLA-binding peptide inhibitor of claim 22, wherein saidinhibitor is further derivatized to reduce the affinity of the analogfor disease-specific T cell receptors.
 24. The HLA-binding peptideinhibitor of claim 22, wherein said inhibitor is further modified toincrease binding potency to an MHC molecule.
 25. A method of treatingCeliac Sprue and/or dermatitis herpetiformis, the method comprising:administering to a patient an effective dose of an HLA-binding peptideinhibitor; wherein said HLA-binding peptide inhibitor attenuates glutentoxicity in said patient.
 26. The method of claim 25, wherein saidHLA-binding peptide inhibitor is administered with a glutenase.
 27. Themethod according to claim 25 wherein said HLA-binding peptide inhibitoris administered orally.
 28. The method according to claim 25, whereinsaid HLA-binding peptide inhibitor is contained in a formulation thatcomprises an enteric coating.
 29. A formulation for use in treatment ofCeliac Sprue and/or dermatitis herpetiformis, comprising: an effectivedose of an HLA-binding peptide inhibitor and a pharmaceuticallyacceptable excipient.
 30. The formulation according to claim 29, furthercomprising an enteric coating.
 31. Use of an HLA-binding peptideinhibitor in the treatment of HLA-DQ2 positive individuals who areeither pre-disposed to type I diabetes or have developed symptoms oftype I diabetes.